Covalent diabodies and uses thereof

ABSTRACT

The present invention is directed to diabody molecules and uses thereof in the treatment of a variety of diseases and disorders, including immunological disorders, infectious disease, intoxication and cancers. The diabody molecules of the invention comprise two polypeptide chains that associate to form at least two epitope binding sites, which may recognize the same or different epitopes on the same or differing antigens. Additionally, the antigens may be from the same or different molecules. The individual polypeptide chains of the diabody molecule may be covalently bound through non-peptide bond covalent bonds, such as, but not limited to, disulfide bonding of cysteine residues located within each polypeptide chain. In particular embodiments, the diabody molecules of the present invention further comprise an Fc region, which allows antibody-like functionality to engineered into the molecule.

This application claims the benefit of U.S. Provisional Application No.60/671,657, filed Apr. 15, 2005, which is hereby incorporated byreference in its entirety.

1. FIELD OF THE INVENTION

The present invention is directed to diabody molecules and uses thereofin the treatment of a variety of diseases and disorders, includingimmunological disorders and cancers. The diabody molecules of theinvention comprise at least two polypeptide chains that associate toform at least two epitope binding sites, which may recognize the same ordifferent epitopes. Additionally, the epitopes may be from the same ordifferent molecules or located on the same or different cells. Theindividual polypeptide chains of the diabody molecule may be covalentlybound through non-peptide bond covalent bonds, such as, but not limitedto, disulfide bonding of cysteine residues located within eachpolypeptide chain. In particular embodiments, the diabody molecules ofthe present invention further comprise an Fc region, which allowsantibody-like functionality to be engineered into the molecule.

2. BACKGROUND OF THE INVENTION

The design of covalent diabodies is based on the single chain Fvconstruct (scFv) (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA90:6444-6448; herein incorporated by reference in its entirety). In anintact, unmodified IgG, the VL and VH domains are located on separatepolypeptide chains, i.e., the light chain and the heavy chain,respectively. Interaction of an antibody light chain and an antibodyheavy chain and, in particular, interaction of VL and VH domains formsone of the epitope binding sites of the antibody. In contrast, the scFvconstruct comprises a VL and VH domain of an antibody contained in asingle polypeptide chain wherein the domains are separated by a flexiblelinker of sufficient length to allow self-assembly of the two domainsinto a functional epitope binding site. Where self assembly of the isimpossible due to a linker of insufficient length (less than about 12amino acid residues), two of the scFv constructs interact with eachother to form a bivalent molecule, the VL of one chain associating withthe VH of the other (reviewed in Marvin et al., 2005, Acta Pharmacol.Sin. 26:649-658). Moreover, addition of a cysteine residue to thec-terminus of the construct has been show to allow disulfide bonding ofthe polypeptide chains, stabilizing the resulting dimer withoutinterfering with the binding characteristics of the bivalent molecule(see e.g., Olafsen et al., 2004, Prot. Engr. Des. Sel. 17:21-27).Further, where VL and VH domains of differing specificity are selected,not only a bivalent, but also a bispecific molecule may be constructed.

Bivalent diabodies have wide ranging applications including therapy andimmunodiagnosis. Bivalency allows for great flexibility in the designand engineering of diabody in various applications, providing enhancedavidity to multimeric antigens, the cross-linking of differing antigens,and directed targeting to specific cell types relying on the presence ofboth target antigens. Due to their increased valency, low dissociationrates and rapid clearance from the circulation (for diabodies of smallsize, at or below ˜50 kDa), diabody molecules known in the art have alsoshown particular use in the filed of tumor imaging (Fitzgerald et al.,1997, Protein Eng. 10:1221). Of particular importance is the crosslinking of differing cells, for example the cross linking of cytotoxic Tcells to tumor cells (Staerz et al., 1985, Nature 314:628-631, andHolliger et al., 1996, Protein Eng. 9:299-305). Diabody epitope bindingdomains may also be directed to a surface determinant of any immuneeffector cell such as CD3, CD16, CD32, or CD64, which are expressed on Tlymphocytes, natural killer (NK) cells or other mononuclear cells. Inmany studies, diabody the effector cell (Holliger et al., 1996, ProteinEng. 9:299-305; Holliger et al., 1999, Cancer Res. 59:2909-2916).Normally, effector cell activation is triggered by the binding of anantigen bound antibody to an effector cell via Fc-FcγR interaction;thus, in this regard, diabody molecules of the invention may exhibitIg-like functionality independent of whether they comprise an Fc domain(e.g., as assayed in any efferctor function assay known in the art orexemplified herein (e.g., ADCC assay)). By cross-linking tumor andeffector cells, the diabody not only brings the effector cell within theproximity of the tumor cells but leads to effective tumor killing (seee.g., Cao and Lam, 2003, Adv. Drug. Deliv. Rev. 55:171-197, herebyincorporated by reference herein in its entirety).

2.1 Effector Cell Receptors and their Roles in the Immune System

In traditional immune function the interaction of antibody-antigencomplexes with cells of the immune system results in a wide array ofresponses, ranging from effector functions such as antibody-dependentcytotoxicity, mast cell degranulation, and phagocytosis toimmunomodulatory signals such as regulating lymphocyte proliferation andantibody secretion. All these interactions are initiated through thebinding of the Fc domain of antibodies or immune complexes tospecialized cell surface receptors on hematopoietic cells. The diversityof cellular responses triggered by antibodies and immune complexesresults from the structural heterogeneity of Fc receptors. Fc receptorsshare structurally related an antigen binding domains which presumablymediate intracellular signaling.

The Fcγ receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fcγ portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe alpha chain of the Fcγ receptor. Fcγ receptors are defined by theirspecificity for immunoglobulin subtypes. Fcγ receptors for IgG arereferred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Differentaccessory cells bear Fcγ receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. 1991,Annu. Rev. Immunol. 9: 457-92; Gerber J. S. et al. 2001 Microbes andInfection, 3: 131-139; Billadeau D. D. et al. 2002, The Journal ofClinical Investigation, 2(109): 161-1681; Ravetch J. V. et al. 2000,Science, 290: 84-89; Ravetch J. V. et al., 2001 Annu. Rev. Immunol.19:275-90; Ravetch J. V. 1994, Cell, 78(4): 553-60). The different Fcγreceptors, the cells that express them, and their isotype specificity issummarized in Table 1 (adapted from Immunobiology: The Immune System inHealth and Disease, 4^(th) ed. 1999, Elsevier Science Ltd/GarlandPublishing, New York).

Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologybetween the three family members suggest they arose from a commonprogenitor perhaps by gene duplication.

FcγRII(CD32)

FcγRII proteins are 40 kDa integral membrane glycoproteins which bindonly the complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹).This receptor is the most widely expressed FcγR, present on allhematopoietic cells, including monocytes, macrophages, B cells, NKcells, neutrophils, mast cells, and platelets. FcγRII has only twoimmunoglobulin-like regions in its immunoglobulin binding chain andhence a much lower affinity for IgG than FcγRI. There are three humanFcγRII genes (FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG inaggregates or immune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A andFcγRII-B create two functionally heterogenous responses to receptorligation. The fundamental difference is that the A isoform initiatesintracellular signaling leading to cell activation such as phagocytosisand respiratory burst, whereas the B isoform initiates inhibitorysignals, e.g., inhibiting B-cell activation.

FcγRIII (CD16)

Due to heterogeneity within this class, the size of FcγRIII rangesbetween 40 and 80 kDa in mouse and man. Two human genes encode twotranscripts, FcγRIIIA, an integral membrane glycoprotein, and FcγRIIIB,a glycosylphosphatidyl-inositol (GPI)-linked version. One murine geneencodes an FcγRIII homologous to the membrane spanning human FcγRIIIA.The FcγRIII shares structural characteristics with each of the other twoFcγRs. Like FcγRII, FcγRIII binds IgG with low affinity and contains thecorresponding two extracellular Ig-like domains. FcγRIIIA is expressedin macrophages, mast cells and is the lone FcγR in NK cells. TheGPI-linked FcγRIIIB is currently known to be expressed only in humanneutrophils.

Signaling Through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called immunoreceptor tyrosine based activation motifs (ITAMs)or immunoreceptor tyrosine based inhibitory motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases which facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofproinflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When co-ligatedalong with an activating FcγR, the ITIM in FcγRIIB becomesphosphorylated and attracts the SH2 domain of the inosital polyphosphate5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengersreleased as a consequence of ITAM-containing FcγR-mediated tyrosinekinase activation, consequently preventing the influx of intracellularCa⁺⁺. Thus crosslinking of FcγRIIB dampens the activating response toFcγR ligation and inhibits cellular responsiveness. B cell activation, Bcell proliferation and antibody secretion is thus aborted. TABLE 1Receptors for the Fc Regions of Immunoglobulin Isotypes FcγRI FcγRII-AFcγRII-B2 FcγRII-B1 FcγRIII FcαRI Receptor (CD64) (CD32) (CD32) (CD32)(CD16) FcεRI (CD89) Binding IgG1 IgG1 IgG1 IgG1 IgG1 IgE IgA1, IgA2 10⁸M⁻¹ 2 × 10⁶ M⁻¹ 2 × 10⁶ M⁻¹ 2 × 10⁶ M⁻¹ 5 × 10⁵ M⁻¹ 1010 M⁻¹ 10⁷ M⁻¹Cell Type Macrophages Macrophages Macrophages B cells NK cells Mastcells Macrophages Neutrophils Neutrophils Neutrophils Mast cellsEosinophil Eosinophil Neutrophils Eosinophils Eosinophils EosinophilsMacrophages Basophils Eosinophils Dendritic cells Dendritic cellsNeutrophils Platelets Mast Cells Langerhan cells Effect of Uptake UptakeUptake No uptake Induction of Secretion of Uptake Ligation StimulationGranule release Inhibition of Inhibition of Killing granules Inductionof Activation of Stimulation Stimulation killing respiratory burstInduction of killing

3. SUMMARY OF THE INVENTION

The present invention relates to covalent diabodies and/or covalentdiabody molecules and to their use in the treatment of a variety ofdiseases and disorders including cancer, autoimmune disorders, allergydisorders and infectious diseases caused by bacteria, fungi or viruses.Preferably, the diabody of the present invention can bind to twodifferent epitopes on two different cells wherein the first epitope isexpressed on a different cell type than the second epitope, such thatthe diabody can bring the two cells together.

In one embodiment, the present invention is directed to a covalentbispecific diabody, which diabody comprises a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope, and,optionally, (iii) a third domain comprising at least one cysteineresidue, which first and second domains are covalently linked such thatthe first and second domains do not associate to form an epitope bindingsite; which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and, optionally, (iii) a sixth domain comprising at least onecysteine residue, which fourth and fifth domains are covalently linkedsuch that the fourth and fifth domains do not associate to form anepitope binding site; and wherein the first polypeptide chain and thesecond polypeptide chain are covalently linked, with the proviso thatthe covalent link is not a peptide bond; wherein the first domain andthe fifth domain associate to form a first binding site (VL1)(VH1) thatbinds the first epitope; wherein the second domain and the fourth domainassociate to form a second binding site (VL2)(VH2) that binds the secondepitope.

In another embodiment, the present invention is directed to a covalentbispecific diabody, which diabody comprises a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope and (iii) athird domain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; which secondpolypeptide chain comprises (i) a fourth domain comprising a bindingregion of a light chain variable domain of the second immunoglobulin(VL2), (ii) a fifth domain comprising a binding region of a heavy chainvariable domain of the first immunoglobulin (VH1), which fourth andfifth domains are covalently linked such that the third and fourthdomains do not associate to form an epitope binding site; and whereinthe first polypeptide chain and the second polypeptide chain arecovalently linked, with the proviso that the covalent link is not apeptide bond; wherein the first domain and the fifth domain associate toform a first binding site (VL1)(VH1) that binds the first epitope;wherein the second domain and the fourth domain associate to form asecond binding site (VL2)(VH2) that binds the second epitope.

In certain aspects, the present invention is directed to diabodymolecule, which molecule comprises a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; which secondpolypeptide chain comprises (i) a fourth domain comprising a bindingregion of a light chain variable domain of the second immunoglobulin(VL2), (ii) a fifth domain comprising a binding region of a heavy chainvariable domain of the first immunoglobulin (VH1), and (iii) a sixthdomain comprising at least one cysteine residue, which fourth and fifthdomains are covalently linked such that the fourth and fifth domains donot associate to form an epitope binding site; and wherein the firstpolypeptide chain and the second polypeptide chain are covalentlylinked, with the proviso that the covalent link is not a peptide bond;wherein the first domain and the fifth domain associate to form a firstbinding site (VL1)(VH1) that binds the first epitope; wherein the seconddomain and the fourth domain associate to form a second binding site(VL2)(VH2) that binds the second epitope.

In certain embodiments, the present invention is directed to a covalentbispecific diabody, which diabody is a dimer of diabody molecules, eachdiabody molecule comprising a first and a second polypeptide chain,which first polypeptide chain comprises (i) a first domain comprising abinding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; and whichsecond polypeptide chain comprises (i) a fourth domain comprising abinding region of a light chain variable domain of the secondimmunoglobulin (VL2), (ii) a fifth domain comprising a binding region ofa heavy chain variable domain of the first immunoglobulin (VH1), and(iii) a sixth domain comprising at least one cysteine residue, whichfourth and fifth domains are covalently linked such that the fourth andfifth domains do not associate to form an epitope binding site; andwherein the first polypeptide chain and the second polypeptide chain ofeach diabody molecule are covalently linked, with the proviso that thecovalent link is not a peptide bond; wherein the first domain and thefifth domain of each diabody molecule associate to form a first bindingsite (VL1)(VH1) that binds the first epitope; wherein the second domainand the fourth domain of each diabody molecule associate to form asecond binding site (VL2)(VH2) that binds the second epitope.

In yet other embodiments, the present invention is directed to acovalent tetrapecific diabody, which diabody is a dimer of diabodymolecules, the first diabody molecule comprising a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope and (iii) athird domain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; and whichsecond polypeptide chain comprises (i) a fourth domain comprising abinding region of a light chain variable domain of the secondimmunoglobulin (VL2), (ii) a fifth domain comprising a binding region ofa heavy chain variable domain of the first immunoglobulin (VH1), and(iii) a sixth domain comprising at least one cysteine residue, whichfourth and fifth domains are covalently linked such that the fourth andfifth domains do not associate to form an epitope binding site; andwherein the first polypeptide chain and the second polypeptide chain arecovalently linked, with the proviso that the covalent link is not apeptide bond; wherein the first domain and the fifth domain associate toform a first binding site (VL1)(VH1) that binds the first epitope;wherein the second domain and the fourth domain associate to form asecond binding site (VL2)(VH2) that binds the second epitope; and thesecond diabody molecule comprising a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a thirdimmunoglobulin (VL3) specific for a third epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a fourthimmunoglobulin (VH4) specific for a fourth epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site; and whichsecond polypeptide chain comprises (i) a fourth domain comprising abinding region of a light chain variable domain of the fourthimmunoglobulin (VL4), (ii) a fifth domain comprising a binding region ofa heavy chain variable domain of the third immunoglobulin (VH3), and(iii) a sixth domain comprising at least one cysteine residue, whichfourth and fifth domains are covalently linked such that the fourth andfifth domains do not associate to form an epitope binding site; andwherein the first polypeptide chain and the second polypeptide chain arecovalently linked, with the proviso that the covalent link is not apeptide bond; wherein the first domain and the fifth domain associate toform a first binding site (VL3)(VH3) that binds the third epitope;wherein the second domain and the fourth domain associate to form asecond binding site (VL4)(VH4) that binds the fourth epitope.

In certain aspects of the invention the first epitope, second epitope,and where applicable, third epitope and fourth epitope can be the same.In other aspects, the first epitope, second epitope, and whereapplicable, third epitope and fourth epitope can each different from theother. In certain aspects of the invention comprising a third epitopebinding domain, the first epitope and third epitope can be the same. Incertain aspects of the invention comprising a fourth epitope bindingdomain, the first epitope and fourth epitope can be the same. In certainaspects of the invention comprising a third epitope binding domain, thesecond epitope and third epitope can be the same. In certain aspects ofthe invention comprising a fourth epitope binding domain, the secondepitope and fourth epitope can be the same. In preferred aspects of theinvention, the first eptitope and second epitope are different. In yetother aspects of the invention comprising a third epitope binding domainand a fourth epitope binding domain, the third epitope and fourthepitope can be different. It is to be understood that any combination ofthe foregoing is encompassed in the present invention.

In particular aspects of the invention, the first domain and the fifthdomain of the diabody or diabody molecule can be derived from the sameimmunoglobulin. In another aspect, the second domain and the fourthdomain of the diabody or diabody molecule can be derived from the sameimmunoglobulin. In yet another aspect, the first domain and the fifthdomain of the diabody or diabody molecule can be derived from adifferent immunoglobulin. In yet another aspect, the second domain andthe fourth domain of the diabody or diabody molecule can be derived froma different immunoglobulin. It is to be understood that any combinationof the foregoing is encompassed in the present invention.

In certain aspects of the invention, the covalent linkage between thefist polypeptide chain and second polypeptide chain of the diabody ordiabody molecule can be via a disulfide bond between at least onecysteine residue on the first polypeptide chain and at least onecysteine residue on the second polypeptide chain. The cysteine residueson the first or second polypeptide chains that are responsible fordisulfide bonding can be found anywhere on the polypeptide chainincluding within the first, second, third, fourth, fifth and sixthdomains. In a specific embodiment the cysteine residue on the firstpolypeptide chain is found in the first domain and the cysteine residueon the second polypeptide chain is found in the fifth domain. The first,second, fourth and fifth domains correspond to the variable regionsresponsible for binding. In preferred embodiments, the cysteine residuesresponsible for the disulfide bonding between the first and secondpolypeptide chains are located within the third and sixth domains,respectively. In a particular aspect of this embodiment, the thirddomain of the first polypeptide chain comprises the C-terminal 6 aminoacids of the human kappa light chain, FNRGEC (SEQ ID NO:23), which canbe encoded by the amino acid sequence (SEQ ID NO:17). In another aspectof this embodiment, the sixth domain of the second polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO:23), which can be encoded by the amino acid sequence(SEQ ID NO:17). In still another aspect of this embodiment, the thirddomain of the first polypeptide chain comprises the amino acid sequenceVEPKSC (SEQ ID NO:79), derived from the hinge domain of a human IgG, andwhich can be encoded by the nucleotide sequence (SEQ ID NO:80). Inanother aspect of this embodiment, the sixth domain of the secondpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ IDNO:79), derived from the hinge domain of a human IgG, and which can beencoded by the nucleotide sequence (SEQ ID NO:80). In certain aspects ofthis embodiment, the third domain of the first polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO:23); and the sixth domain of the second polypeptidechain comprises the amino acid sequence VEPKSC (SEQ ID NO:79). In otheraspects of this embodiment, the sixth domain of the second polypeptidechain comprises the C-terminal 6 amino acids of the human kappa lightchain, FNRGEC (SEQ ID NO:23); and the third domain of the firstpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ IDNO:79). In yet other aspects of this embodiment, the third domain of thefirst polypeptide chain comprises the C-terminal 6 amino acids of thehuman kappa light chain, FNRGEC (SEQ ID NO:23); and the sixth domain ofthe second polypeptide chain comprises a hinge domain. In other aspectsof this embodiment, the sixth domain of the second polypeptide chaincomprises the C-terminal 6 amino acids of the human kappa light chain,FNRGEC (SEQ ID NO:23); and the third domain of the first polypeptidechain comprises the hinge domain. In yet other aspects of thisembodiment, the third domain of the first polypeptide chain comprisesthe C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQID NO:23); and the sixth domain of the first polypeptide chain comprisesan Fc domain, or portion thereof. In still other aspects of thisembodiment, the sixth domain of the second polypeptide chain comprisesthe C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQID NO:23); and the third domain of the first polypeptide chain comprisesan Fc domain, or portion thereof.

In other embodiments, the cysteine residues on the first or secondpolypeptide that are responsible for the disulfide bonding can belocated outside of the first, second or third domains on the firstpolypeptide chain and outside of the fourth, fifth and sixth domain onthe second polypeptide chain. In particular, the cysteine residue on thefirst polypeptide chain can be N-terminal to the first domain or can beC-terminal to the first domain. The cysteine residue on the firstpolypeptide chain can be N-terminal to the second domain or can beC-terminal to the second domain. The cysteine residue on the firstpolypeptide chain can be N-terminal to the third domain or can beC-terminal to the third domain. Further, the cysteine residue on thesecond polypeptide chain can be N-terminal to the fourth domain or canbe C-terminal to the fourth domain. The cysteine residue on the secondpolypeptide chain can be N-terminal to the fifth domain or can beC-terminal to the fifth domain. Accordingly, the cysteine residue on thesecond polypeptide chain can be C-terminal to the sixth domain or can beN-terminal to the sixth domain. In a particular aspect, disulfide bondcan between at least two cysteine residues on the first polypeptidechain and at least two cysteine residues on the second polypeptidechain. In a particular aspect, wherein the third domain and sixth domaindo not comprise an Fc domain, or portion thereof, the cysteine residuecan be at the C-terminus of the first polypeptide chain and at theC-terminus of the second polypeptide chain. It is to be understood thatany combination of the foregoing is encompassed in the presentinvention.

In specific embodiments of the invention described supra, the covalentdiabody of the invention encompasses dimers of diabody molecules,wherein each diabody molecule comprises a first and second polypeptidechain. In certain aspects of this embodiment the diabody molecules canbe covalently linked to form the dimer, with the proviso that thecovalent linkage is not a peptide bond. In preferred aspects of thisembodiment, the covalent linkage is a disulfide bond between at leastone cysteine residue on the first polypeptide chain of each of thediabody molecules of the dimer. In yet more preferred aspects of thisinvention, the covalent linkage is a disulfide bond between at least onecysteine residue on the first polypeptide chain of each of the diabodymolecules forming the dimer, wherein said at least one cysteine residueis located in the third domain of each first polypeptide chain.

In certain aspects of the invention, the first domain on the firstpolypeptide chain can be N-terminal to the second domain or can beC-terminal to the second domain. The first domain on the firstpolypeptide chain can be N-terminal to the third domain or can beC-terminal to the third domain. The second domain on the firstpolypeptide chain can be N-terminal to the first domain or can beC-terminal to the first domain. Further, the second domain on the firstpolypeptide chain can be N-terminal to the third domain or can beC-terminal to the third domain. Accordingly, the third domain on thefirst polypeptide chain can be N-terminal to the first domain or can beC-terminal to the first domain. The third domain on the firstpolypeptide chain can be N-terminal to the second domain or can beC-terminal to the second domain. With respect to the second polypeptidechain, the fourth domain can be N-terminal to the fifth domain or can beC-terminal to the fifth domain. The fourth domain can be N-terminal tothe sixth domain or can be C-terminal to the sixth domain. The fifthdomain on the second polypeptide chain can be N-terminal to the fourthdomain or can be C-terminal to the fourth domain. The fifth domain onthe second polypeptide chain can be N-terminal to the sixth domain orcan be C-terminal to the sixth domain. Accordingly the sixth domain onthe second polypeptide chain can be N-terminal to the fourth domain orcan be C-terminal to the fourth domain. The sixth domain on the secondpolypeptide chain can be N-terminal to the fifth domain or can beC-terminal to the fifth domain. It is to be understood that anycombination of the foregoing is encompassed in the present invention.

In certain embodiments, first domain and second domain can be locatedC-terminal to the third domain on the first polypeptide chain; or thefirst domain and second domain can be located N-terminal to the thirddomain on the first polypeptide chain. With respect to the secondpolypeptide chain, the fourth domain and fifth domain can be locatedC-terminal to the sixth domain, or the fourth domain and fifth domaincan be located N-terminal to the sixth domain. In certain aspects ofthis embodiment, the present invention is directed to a covalentbispecific diabody, which diabody is a dimer of diabody molecules, eachdiabody molecule comprising a first and a second polypeptide chain,which first polypeptide chain comprises (i) a first domain comprising abinding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site and wherein thethird domain is located N-terminal to both the first domain and seconddomain; and which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and (iii) a sixth domain comprising at least one cysteineresidue, which fourth and fifth domains are covalently linked such thatthe fourth and fifth domains do not associate to form an epitope bindingsite; and wherein the first polypeptide chain and the second polypeptidechain of each diabody molecule are covalently linked, with the provisothat the covalent link is not a peptide bond; wherein the first domainand the fifth domain of each diabody molecule associate to form a firstbinding site (VL1)(VH1) that binds the first epitope; wherein the seconddomain and the fourth domain of each diabody molecule associate to forma second binding site (VL2)(VH2) that binds the second epitope.

In yet another embodiment, the present invention is directed to acovalent tetrapecific diabody, which diabody is a dimer of diabodymolecules, the first diabody molecule comprising a first and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope and (iii) athird domain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site and wherein thethird domain is located N-terminal to both the first domain and seconddomain; and which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and (iii) a sixth domain comprising at least one cysteineresidue, which fourth and fifth domains are covalently linked such thatthe fourth and fifth domains do not associate to form an epitope bindingsite; and wherein the first polypeptide chain and the second polypeptidechain are covalently linked, with the proviso that the covalent link isnot a peptide bond; wherein the first domain and the fifth domainassociate to form a first binding site (VL1)(VH1) that binds the firstepitope; wherein the second domain and the fourth domain associate toform a second binding site (VL2)(VH2) that binds the second epitope; andthe second diabody molecule comprises a first and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a thirdimmunoglobulin (VL3) specific for a third epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a fourthimmunoglobulin (VH4) specific for a fourth epitope and (iii) a thirddomain comprising an Fc domain or portion thereof, which first andsecond domains are covalently linked such that the first and seconddomains do not associate to form an epitope binding site and wherein thethird domain is located N-terminal to both the first domain and seconddomain; and which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thefourth immunoglobulin (VL4), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the third immunoglobulin(VH3), and (iii) a sixth domain comprising at least one cysteineresidue, which fourth and fifth domains are covalently linked such thatthe fourth and fifth domains do not associate to form an epitope bindingsite; and wherein the first polypeptide chain and the second polypeptidechain are covalently linked, with the proviso that the covalent link isnot a peptide bond; wherein the first domain and the fifth domainassociate to form a first binding site (VL3)(VH3) that binds the thirdepitope; wherein the second domain and the fourth domain associate toform a second binding site (VL4)(VH4) that binds the fourth epitope.

As discussed above, the domains on the individual polypeptide chains arecovalently linked. In specific aspects, the covalent link between thefirst and second domain, first and third domain, second and thirddomain, fourth and fifth domain, fourth and sixth domain, and/or fifthand sixth domain can be a peptide bond. In particular, the first andsecond domains, and the fourth and fifth domains can be separated by thethird domain and sixth domain, respectively, or by additional amino acidresidues, so long as the first and second, and fourth and fifth domainsdo not associate to form a binding site. The number of amino acidresidues can be 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid residues. Inone preferred aspect, the number of amino acid residues between thedomains is 8.

In certain aspects of the invention, the domains of the first and secondpolypeptid chain comprising an Fc domain, i.e., optionally, the thirdand sixth domains, respectively, can further comprise a hinge domainsuch that the domain comprises a hinge-Fc region. In alternativeembodiments, the first polypeptide chain or the second polypeptide chaincan comprise a hinge domain without also comprising an Fc domain. Theheavy chains, light chains, hinge regions, Fc domains, and/or hinge-Fcdomains for use in the invention can be derived from any immunoglobulintype including IgA, IgD, IgE, IgG or IgM. In a preferred aspect, theimmunoglobulin type is IgG, or any subtype thereof, i.e., IgG₁, IgG₂,IgG₃ or IgG₄. In other aspects, the immunoglobulin from which the lightand heavy chains are derived is humanized or chimerized.

Further, the first epitope and second epitopes, and, where applicable,third epitope and fourth epitope, to which the diabody or diabodymolecule binds can be different epitopes from the same antigen or can bedifferent epitopes from different antigens. The antigens can be anymolecule to which an antibody can be generated. For example, proteins,nucleic acids, bacterial toxins, cell surface markers, autoimmunemarkers, viral proteins, drugs, etc. In particular aspects, at least oneepitope binding site of the diabody is specific for an antigen on aparticular cell, such as a B-cell, a T-cell, a phagocytic cell, anatural killer (NK) cell or a dendritic cell.

In certain aspects of the present embodiment, at least one epitopebinding site of the diabody or diabody molecule is specific for a Fcreceptor, which Fc receptor can be an activating Fc receptor or aninhibitory Fc receptor. In particular aspects, the Fc receptor is a Fcγreceptor, and the Fcγ receptor is a FcγRI, FcγRII or FcγRIII receptor.In more preferred aspects, the FcγRIII receptor is the FcγRIIIA (CD16A)receptor or the FcγRIIIB (CD16B) receptor, and, more preferably, theFcγRIII receptor is the FcγRIIIA (CD16A) receptor. In another preferredaspect, the FcγRII receptor is the FcγRIIA (CD32A) receptor or theFcγRIIB (CD32B) receptor, and more preferably the FcγRIIB (CD32B)receptor. In a particularly preferred aspect, one binding site of thediabody is specific for CD32B and the other binding site is specific forCD16A. In a specific embodiment of the invention, at least one epitopebinding site of the diabody or diabody molecule is specific for anactivating Fc receptor and at least one other site is specific for aninhibitory Fc receptor. In certain aspects of this embodiment theactivating Fc receptor is CD32A and the inhibitory Fc receptor is CD32B.In other aspects of this embodiment the activating Fc receptor is BCRand the inhibitory Fc receptor is CD32B. In still other aspects of thisembodiment, the activating Fc receptor is IgERI and the inhibitory Fcreceptor is CD32B.

In cases where one epitope binding site is specific for CD16A, the VLand VH domains can be the same as or similar to the VL and VH domains ofthe mouse antibody 3G8, the sequence of which has been cloned and is setforth herein. In other cases where one epitope binding site is specificfor CD32A, the VL and VH domains can be the same as or similar to the VLand VH domains of the mouse antibody IV.3. In yet other cases where oneepitope binding site is specific for CD32B, the VL and VH domains can bethe same as or similar to the VL and VH domains of the mouse antibody2B6, the sequence of which has been cloned and is set forth herein. Itis to be understood that any of the VL or VH domains of the 3G8, 2B6 andIV.3 antibodies can be used in any combination. The present invention isalso directed to a bispecific diabody or diabody molecule wherein thefirst epitope is specific for CD32B, and the second epitope is specificfor CD16A.

In other aspects, an epitope binding site can be specific for apathogenic antigen. As used herein, a pathogenic antigen is an antigeninvolved in a specific pathogenic disease, including cancer, infectionand autoimmune disease. Thus, the pathogenic antigen can be a tumorantigen, a bacterial antigen, a viral antigen, or an autoimmune antigen.Exemplary pathogenic antigens include, but are not limited tolipopolysaccharide, viral antigens selected from the group consisting ofviral antigens from human immunodeficiency virus, Adenovirus,Respiratory Syncitial Virus, West Nile Virus (e.g., E16 and/or E53antigens) and hepatitis virus, nucleic acids (DNA and RNA) and collagen.Preferably, the pathogenic antigen is a neutralizing antigen. In apreferred aspect, where one epitope binding site is specific for CD16Aor CD32A, the other epitope binding site is specific for a pathogenicantigen excluding autoimmune antigens. In yet another preferred aspect,where one epitope binding site is specific for CD32B, the other epitopebinding site is specific for any pathogenic antigen. In specificembodiments, the diabody molecule of the invention binds two differentantigen on the same cell, for example, one antigen binding site isspecific for an activating Fc receptor while the other is specific foran inhibitory Fc receptor. In other embodiments, the diabody moleculebinds two distinct viral neutralizing epitopes, for example, but notlimited to, E16 and E53 of West Nile Virus.

In yet another embodiment of the present invention, the diabodies of theinvention can be used to treat a variety of diseases and disorders.Accordingly, the present invention is directed to a method for treatinga disease or disorder comprising administering to a patient in needthereof an effective amount of a covalent diabody or diabody molecule ofthe invention in which at least one binding site is specific for apathogenic antigen, such as an antigen expressed on the surface of acancer cell or on the surface of a bacterium or virion and at least oneother binding site is specific for a Fc receptor, e.g., CD16A.

In yet another embodiment, the invention is directed to a method fortreating a disease or disorder comprising administering to a patient inneed thereof an effective amount of a diabody or diabody molecule of theinvention, in which at least one binding site is specific for CD32B andat least one other binding site is specific for CD16A.

In yet another embodiment, the invention is directed to a method forinducing immune tolerance to a pathogenic antigen comprisingadministering to a patient in need there an effective amount of acovalent diabody or dovalent diabody molecule of the invention, in whichat least one binding site is specific for CD32B and at least one otherbinding site is specific for said pathogenic antigen. In aspects of thisembodiment, the pathogenic antigen can be an allergen or anothermolecule to which immune tolerance is desired, such as a proteinexpressed on transplanted tissue.

In yet another embodiment, the present invention is directed to a methodfor detoxification comprising administering to a patient in need thereofan effective amount of a covalent diabody or diabody molecule of theinvention, in which at least one binding site is specific for a cellsurface marker and at least one other binding site is specific for atoxin. In particular aspects, the diabody of the invention administeredis one where one binding site is specific for a cell surface marker suchas an Fc and the other binding site is specific for a bacterial toxin orfor a drug. In one aspect, the cell surface marker is not found on redblood cells.

3.1 Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. The practice of the present invention willemploy, unless otherwise indicated, conventional techniques of molecularbiology (including recombinant techniques), microbiology, cell biology,biochemistry, nucleic acid chemistry, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Current Protocols in Immunology (J. E. Coligan etal., eds., 1999, including supplements through 2001); Current Protocolsin Molecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); Molecular Cloning: A Laboratory Manual, thirdedition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); The Immunoassay Handbook (D. Wild, ed.,Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson,ed., Academic Press, 1996); Methods of Immunological Analysis (R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlagsgesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1999; and Beaucage et al. eds., Current Protocols in Nucleic AcidChemistry John Wiley & Sons, Inc., New York, 2000).

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv),single chain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic(anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Idantibodies to antibodies of the invention), and epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁,and IgA₂) or subclass.

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” “specificallyrecognizes” and analogous terms refer to molecules that specificallybind to an antigen (e.g., eptiope or immune complex) and do notspecifically bind to another molecule. A molecule that specificallybinds to an antigen may bind to other peptides or polypeptides withlower affinity as determined by, e.g., immunoassays, BIAcore, or otherassays known in the art. Preferably, molecules that specifically bind anantigen do not cross-react with other proteins. Molecules thatspecifically bind an antigen can be identified, for example, byimmunoassays, BIAcore, or other techniques known to those of skill inthe art.

As used herein, immune complex, refers to a structure which forms whenat least one target molecule and at least one heterologous Fcγregion-containing polypeptide bind to one another forming a largermolecular weight complex. Examples of immune complexes areantigen-antibody complexes which can be either soluble or particulate(e.g., an antigen/antibody complex on a cell surface.).

As used herein, the terms “heavy chain,” “light chain,” “variableregion,” “framework region,” “constant domain,” and the like, have theirordinary meaning in the immunology art and refer to domains in naturallyoccurring immunoglobulins and the corresponding domains of synthetic(e.g., recombinant) binding proteins (e.g., humanized antibodies, singlechain antibodies, chimeric antibodies, etc.). The basic structural unitof naturally occurring immunoglobulins (e.g., IgG) is a tetramer havingtwo light chains and two heavy chains, usually expressed as aglycoprotein of about 150,000 Da. The amino-terminal (“N”) portion ofeach chain includes a variable region of about 100 to 110 or more aminoacids primarily responsible for antigen recognition. Thecarboxy-terminal (“C”) portion of each chain defines a constant region,with light chains having a single constant domain and heavy chainsusually having three constant domains and a hinge region. Thus, thestructure of the light chains of an IgG molecule is n-V_(L)—C_(L)-c andthe structure of IgG heavy chains is n-V_(H)—C_(H1)—H—C_(H2)—C_(H3)-c(where H is the hinge region). The variable regions of an IgG moleculeconsist of the complementarity determining regions (CDRs), which containthe residues in contact with antigen and non-CDR segments, referred toas framework segments, which in general maintain the structure anddetermine the positioning of the CDR loops (although certain frameworkresidues may also contact antigen). Thus, the V_(L) and V_(H) domainshave the structure n-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4-c.

When referring to binding proteins or antibodies (as broadly definedherein), the assignment of amino acids to each domain is in accordancewith the definitions of Kabat, Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md., 1987 and 1991).Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated by the position of an aminoacid in the chain. Kabat described numerous amino acid sequences forantibodies, identified an amino acid consensus sequence for eachsubgroup, and assigned a residue number to each amino acid. Kabat'snumbering scheme is extendible to antibodies not included in hiscompendium by aligning the antibody in question with one of theconsensus sequences in Kabat by reference to conserved amino acids. Thismethod for assigning residue numbers has become standard in the fieldand readily identifies amino acids at equivalent positions in differentantibodies, including chimeric or humanized variants. For example, anamino acid at position 50 of a human antibody light chain occupies theequivalent position to an amino acid at position 50 of a mouse antibodylight chain.

As used herein, the term “heavy chain” is used to define the heavy chainof an IgG antibody. In an intact, native IgG, the heavy chain comprisesthe immunoglobulin domains VH, CH1, hinge, CH2 and CH3. Throughout thepresent specification, the numbering of the residues in an IgG heavychain is that of the EU index as in Kabat et al., Sequences of Proteinsof Immunological Interest, 5^(th) Ed. Public Health Service, NH1, MD(1991), expressly incorporated herein by references. The “EU index as inKabat” refers to the numbering of the human IgG1 EU antibody. Examplesof the amino acid sequences containing human IgG1 hinge, CH2 and CH3domains are shown in FIGS. 1A and 1B as described, infra. FIGS. 1A and1B also set forth amino acid sequences of the hinge, CH2 and CH3 domainsof the heavy chains of IgG2, IgG3 and IgG4. The amino acid sequences ofIgG2, IgG3 and IgG4 isotypes are aligned with the IgG1 sequence byplacing the first and last cysteine residues of the respective hingeregions, which form the inter-heavy chain S—S bonds, in the samepositions. For the IgG2 and IgG3 hinge region, not all residues arenumbered by the Eu index.

The “hinge region” or “hinge domain” is generally defined as stretchingfrom Glu216 to Pro230 of human IgG1. An example of the amino acidsequence of the human IgG1 hinge region is shown in FIG. 1A (amino acidresidues in FIG. 1A are numbered according to the Kabat system). Hingeregions of other IgG isotypes may be aligned with the IgG1 sequence byplacing the first and last cysteine residues forming inter-heavy chainS—S binds in the same positions as shown in FIG. 1A.

As used herein, the term “Fc region,” “Fc domain” or analogous terms areused to define a C-terminal region of an IgG heavy chain. An example ofthe amino acid sequence containing the human IgG1 is shown in FIG. 1B.Although boundaries may vary slightly, as numbered according to theKabat system, the Fc domain extends from amino acid 231 to amino acid447 (amino acid residues in FIG. 1B are numbered according to the Kabatsystem). FIG. 1B also provides examples of the amino acid sequences ofthe Fc regions of IgG isotypes IgG2, IgG3, and IgG4.

The Fc region of an IgG comprises two constant domains, CH2 and CH3. TheCH2 domain of a human IgG Fc region usually extends from amino acids 231to amino acid 341 according to the numbering system of Kabat (FIG. 1B).The CH3 domain of a human IgG Fc region usually extends from amino acids342 to 447 according to the numbering system of Kabat (FIG. 1B). The CH2domain of a human IgG Fc region (also referred to as “Cγ2” domain) isunique in that it is not closely paired with another domain. Rather, twoN-linked branched carbohydrate chains are interposed between the two CH2domains of an intact native IgG.

As used herein the terms “FcγR binding protein,” “FcγR antibody,” and“anti-FcγR antibody”, are used interchangeably and refer to a variety ofimmunoglobulin-like or immunoglobulin-derived proteins. “FcγR bindingproteins” bind FcγR via an interaction with V_(L) and/or V_(H) domains(as distinct from Fcγ-mediated binding). Examples of FcγR bindingproteins include fully human, polyclonal, chimeric and humanizedantibodies (e.g., comprising 2 heavy and 2 light chains), fragmentsthereof (e.g., Fab, Fab′, F(ab′)₂, and Fv fragments), bifunctional ormultifunctional antibodies (see, e.g., Lanzavecchia et al., 1987, Eur.J. Immunol. 17:105), single chain antibodies (see, e.g., Bird et al.,1988, Science 242:423-26), fusion proteins (e.g., phage display fusionproteins), “minibodies” (see, e.g., U.S. Pat. No. 5,837,821) and otherantigen binding proteins comprising a V_(L) and/or V_(H) domain orfragment thereof. In one aspect, the FcγRIIIA binding protein is a“tetrameric antibody” i.e., having generally the structure of anaturally occurring IgG and comprising variable and constant domains,i.e., two light chains comprising a V_(L) domain and a light chainconstant domain and two heavy chains comprising a V_(H) domain and aheavy chain hinge and constant domains.

As used herein the term “FcγR antagonists” and analogous terms refer toprotein and non-proteinacious substances, including small moleculeswhich antagonize at least one biological activity of an FcγR, e.g.,block signaling. For example, the molecules of the invention blocksignaling by blocking the binding of IgGs to an FcγR.

As used herein, the term “derivative” in the context of polypeptides orproteins refers to a polypeptide or protein that comprises an amino acidsequence which has been altered by the introduction of amino acidresidue substitutions, deletions or additions. The term “derivative” asused herein also refers to a polypeptide or protein which has beenmodified, i.e, by the covalent attachment of any type of molecule to thepolypeptide or protein. For example, but not by way of limitation, anantibody may be modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularan antigen or other protein, etc. A derivative polypeptide or proteinmay be produced by chemical modifications using techniques known tothose of skill in the art, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Further, a derivative polypeptide or proteinderivative possesses a similar or identical function as the polypeptideor protein from which it was derived.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative refers to a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated.

As used herein, the term “diabody molecule” refers to a complex of twoor more polypeptide chains or proteins, each comprising at least one VLand one VH domain or fragment thereof, wherein both domains arecomprised within a single polypeptide chain. In certain embodiments“diabody molecule” includes molecules comprising an Fc or a hinge-Fcdomain. Said polypeptide chains in the complex may be the same ordifferent, i.e., the diabody molecule may be a homo-multimer or ahetero-multimer. In specific aspects, “diabody molecule” includes dimersor tetramers or said polypeptide chains containing both a VL and VHdomain. The individual polypeptide chains comprising the multimericproteins may be covalently joined to at least one other peptide of themultimer by interchain disulfide bonds.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

“Identical polypeptide chains” as used herein also refers to polypeptidechains having almost identical amino acid sequence, for example,including chains having one or more amino acid differences, preferablyconservative amino acid substitutions, such that the activity of the twopolypeptide chains is not significantly different

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. In someembodiments, cancer refers to a benign tumor, which has remainedlocalized. In other embodiments, cancer refers to a malignant tumor,which has invaded and destroyed neighboring body structures and spreadto distant sites. In some embodiments, the cancer is associated with aspecific cancer antigen.

As used herein, the term “immunomodulatory agent” and variations thereofrefer to an agent that modulates a host's immune system. In certainembodiments, an immunomodulatory agent is an immunosuppressant agent. Incertain other embodiments, an immunomodulatory agent is animmunostimulatory agent. Immunomodatory agents include, but are notlimited to, small molecules, peptides, polypeptides, fusion proteins,antibodies, inorganic molecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to a fragment of a polypeptideor protein or a non-protein molecule having antigenic or immunogenicactivity in an animal, preferably in a mammal, and most preferably in ahuman. An epitope having immunogenic activity is a fragment of apolypeptide or protein that elicits an antibody response in an animal.An epitope having antigenic activity is a fragment of a polypeptide orprotein to which an antibody immunospecifically binds as determined byany method well-known to one of skill in the art, for example byimmunoassays. Antigenic epitopes need not necessarily be immunogenic.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder. A therapeutically effective amount may refer to the amountof therapeutic agent sufficient to delay or minimize the onset ofdisease, e.g., delay or minimize the spread of cancer. A therapeuticallyeffective amount may also refer to the amount of the therapeutic agentthat provides a therapeutic benefit in the treatment or management of adisease. Further, a therapeutically effective amount with respect to atherapeutic agent of the invention means the amount of therapeutic agentalone, or in combination with other therapies, that provides atherapeutic benefit in the treatment or management of a disease.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence or onset of one or more symptoms ofa disorder in a subject as result of the administration of aprophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder. Afirst prophylactic or therapeutic agent can be administered prior to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) theadministration of a second prophylactic or therapeutic agent to asubject with a disorder.

“Effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or an antigen. Effector functions include but are not limitedto antibody dependent cell mediated cytotoxicity (ADCC), antibodydependent cell mediated phagocytosis (ADCP), and complement dependentcytotoxicity (CDC). Effector functions include both those that operateafter the binding of an antigen and those that operate independent ofantigen binding.

“Effector cell” as used herein is meant a cell of the immune system thatexpresses one or more Fc receptors and mediates one or more effectorfunctions. Effector cells include but are not limited to monocytes,macrophages, neutrophils, dendritic cells, eosinophils, mast cells,platelets, B cells, large granular lymphocytes, Langerhans' cells,natural killer (NK) cells, and may be from any organism including butnot limited to humans, mice, rats, rabbits, and monkeys.

As used herein, the term “specifically binds an immune complex” andanalogous terms refer to molecules that specifically bind to an immunecomplex and do not specifically bind to another molecule. A moleculethat specifically binds to an immune complex may bind to other peptidesor polypeptides with lower affinity as determined by, e.g.,immunoassays, BIAcore, or other assays known in the art. Preferably,molecules that specifically bind an immune complex do not cross-reactwith other proteins. Molecules that specifically bind an immune complexcan be identified, for example, by immunoassays, BIAcore, or othertechniques known to those of skill in the art.

A “stable fusion protein” as used herein refers to a fusion protein thatundergoes minimal to no detectable level of degradation duringproduction and/or storage as assessed using common biochemical andfunctional assays known to one skilled in the art, and can be stored foran extended period of time with no loss in biological activity, e.g.,binding to FcγR.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B Amino Acid Sequence of Human IgG CH1, Hinge and Fc Regions

FIG. 1 provides the amino acid sequences of human IgG1, IgG2, IgG3 andIgG4 hinge (A) and Fc (B) domains. (IgG1 hinge domain (SEQ ID NO:1);IgG2 hinge domain (SEQ ID NO:2); IgG3 hinge domain (SEQ ID NO:3); IgG4hinge domain (SEQ ID NO:4); IgG1 Fc domain (SEQ ID NO:5); IgG2 Fc domain(SEQ ID NO:6); IgG3 Fc domain (SEQ ID NO:7); IgG1 Fc domain (SEQ IDNO:8)). The amino acid residues shown in FIGS. 1A and 1B are numberedaccording to the numbering system of Kabat EU. Isotype sequences arealigned with the IgG1 sequence by placing the first and last cysteineresidues of the respective hinge regions, which form the inter-heavychain S—S bonds, in the same positions. For figure 1B, residues in theCH2 domain are indicated by +, while residues in the CH3 domain areindicated by ˜.

FIG. 2 Schematic Representation of Polypeptide Chains of CovalentBifunctional Diabodies

Polypeptides of a covalent, bifunctional diabody consist of an antibodyVL and an antibody VH domain separated by a short peptide linker. The 8amino acid residue linker prevents self assembly of a single polypeptidechain into scFv constructs, and, instead, interactions between the VLand VH domains of differing polypeptide chains predominate. 4 constructswere created (each construct is described from the amino terminus (“n”),left side of the construct, to the carboxy terminus (“c”), right side offigure): construct (1) (SEQ ID NO:9) comprised, n—the VL domainHu2B6—linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu3G8—and aC-terminal sequence (LGGC)-c; construct (2) (SEQ ID NO:11) comprisedn—the VL domain Hu3G8—linker (GGGSGGGG (SEQ ID NO:10))—the VH domain ofHu2B6—and a C-terminal sequence (LGGC)-c; construct (3) (SEQ ID NO:12)comprised n—the VL domain Hu3G8—linker (GGGSGGGG (SEQ ID NO:10))—the VHdomain of Hu3G8—and a C-terminal sequence (LGGC)-c; construct (4) (SEQID NO:13) comprised n—the VL domain Hu2B6—linker (GGGSGGGG (SEQ IDNO:10))—the VH domain of Hu2B6—and a C-terminal sequence (LGGC)-c.

FIG. 3 SDS-PAGE Analysis of Affinity Purified Diabodies

Affinity purified diabodies were subjected to SDS-PAGE analysis underreducing (lanes 1-3) or non-reducing (lanes 4-6) conditions. Approximatemolecular weights of the standard (in between lanes 3 and 4) areindicated. Lanes 1 and 4, h3G8 CMD; Lanes 2 and 5, h2B6 CMD; and Lanes 3and 6, h2B6-h3G8 CBD.

FIGS. 4A-B SEC Analysis of Affinity Purified Diabodies

Affinity purified diabodies were subjected to SEC analysis. (A) Elutionprofile of known standards: full-length IgG (˜150 kDa), Fab fragment ofIgG (˜50 kDa), and scFv (˜30 kDa); (B) Elution profile of h2b6 CMD, h3G8CMD, and h2B6-h3G8 CBD.

FIG. 5 Binding of h2B6-h3G8 CBD to sCD32B and sCD16A

The binding of h2B6-h3G8 CBD to sCD32B and sCD16A was assayed in asandwich ELISA. sCD32B was used as the target protein. The secondaryprobe was HRP conjugated sCD16A. h3G8 CMD, which binds CD16A, was usedas control.

FIGS. 6A-C Biacore Analysis of Diabody Binding to sCD16A, sCD32B andsCD32B

The binding of h2B6-h3G8 CBD, h2B6 CMD and h3G8 CMD to sCD16A, sCD32B,and sCD32A (negative control) was assayed by SPR analysis. h3G8 scFv wasalso tested as a control. (A) Binding to sCD16; (B) Binding to sCD32Band (C) Binding to sCD32A. Diabodies were injected at a concentration of100 NM, and scFv at a concentration of 200 nM, over receptor surfaces ata flow rate of 50 ml/min for 60 sec.

FIGS. 7A-C Biacore Analysis of Diabody Binding to sCD16A and sCD32B

The binding of h2B6-h3G8 CBD, h2B6 CMD and h3G8 CMD to sCD16A, andsCD32B was assayed by SPR analysis. h3G8 scFv was also tested as acontrol. (A) Binding of to h3G8 CMD sCD16A; (B) Binding of h2B6-h3G8 CBDto sCD16A; (C) Binding of h3G8 scFv to sCD16A; (D) Binding of h2B6 CMDto sCD32B; and (E) Binding of h2B6-h3G8 CBD to sCD32B. Diabodies wereinjected at concentrations of 6.25-200 nM over receptor surfaces at aflow rate of 70 ml/min for 180 sec.

FIG. 8 Schematic Depicting the Interaction of Polypeptide ChainsComprising VL and VH Domains to Form a Covalent Bispecific DiabodyMolecule

NH₂ and COOH represent the amino-terminus and carboxy terminus,respectively of each polypeptide chain. S represents the C-terminalcysteine residue on each polypeptide chain. VL and VH indicate thevariable light domain and variable heavy domain, respectively. Dottedand dashed lines are to distinguish between the two polypeptide chainsand, in particular, represent the linker portions of said chains. h2B6Fv and h3G8 Fv indicate an epitope binding site specific for CD32B andCD16, respectively.

FIG. 9 Schematic Representation of Polypeptide Chains Containing FcDomains of Covalent Bispecific Diabodies

Representation of polypeptide constructs of the diabody molecules of theinvention (each construct is described from the amino terminus (“n”),left side of the construct, to the carboxy terminus (“c”), right side offigure). Construct (5) (SEQ ID NO:14) comprised, n—VL domain Hu2B6—afirst linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu3G8—a secondlinker (LGGC)- and a C-terminal Fc domain of human IgG1-c; construct (6)(SEQ ID NO:15) comprised n—the VL domain Hu3G8—linker (GGGSGGGG (SEQ IDNO:10))—the VH domain of Hu2B6—and second linker (LGGC)- and aC-terminal Fc domain of human IgG1-c; construct (7) (SEQ ID NO:16)comprised n—the VL domain Hu2B6—a first linker (GGGSGGGG (SEQ IDNO:10))—the VH domain of Hu3G8—and a C-terminal sequence (LGGCFNRGEC)(SEQ ID NO:17)-c; construct (8) (SEQ ID NO:18) comprised n—the VL domainHu3G8—linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu2B6—and secondlinker (LGGC)- and a C-terminal hinge/Fc domain of human IgG1 (withamino acid substitution A215V)-c.

FIG. 10 Binding of Diabody Molecules Comprising Fc Domains to sCD32B andsCD16A

The binding of diabody molecules comprising Fc domains to sCD32B andsCD16A was assayed in a sandwich ELISA. Diabodies assayed were producedby 3 recombinant expression systems: cotransfection of pMGX669 andpMGX674, expressing constructs 1 and 6, respectively; cotransfection ofpMGX667 and pMGX676, expressing constructs 2 and 5, respectively; andcotransfection of pMGX674 and pMGX676, expressing constructs 5 and 6,respectively. sCD32B was used as the target protein. The secondary probewas HRP conjugated sCD16A.

FIG. 11 Schematic Depicting the Interaction of Two Polypeptide ChainsEach Comprising an Fc Domain to Form a Bivalent, Covalent Diabody

NH₂ and COOH represent the amino-terminus and carboxy terminus,respectively of each polypeptide chain. S represents the at least onedisulfide bond between a cysteine residue in the second linker sequenceof each polypeptide chain. VL and VH indicate the variable light domainand variable heavy domain, respectively. Dotted and dashed lines are todistinguish between the two polypeptide chains and, in particular,represent the first linker portions of said chains. CH2 and CH3represent the CH2 and CH3 constant domains of an Fc domain. h2B6 Fv andh3G8 Fv indicate an epitope binding site specific for CD32B and CD16,respectively.

FIG. 12 Binding of Diabody Molecules Comprising Hinge/Fc Domains tosCD32B and sCD16A

The binding of diabody molecules comprising Fc domains to sCD32B andsCD16A was assayed in a sandwich ELISA. Diabodies assayed were producedby 4 recombinant expression systems: cotransfection of pMGX669+pMGX674,expressing constructs 1 and 6, respectively; cotransfection ofpMGX669+pMGX678, expressing constructs 2 and 8, respectively;cotransfection of pMGX677+pMGX674, expressing constructs 7 and 6,respectively; and cotransfection of pMGX677+pMGX678, expressingconstructs 7 and 8, respectively. sCD32B was used as the target protein.The secondary probe was HRP conjugated sCD16A.

FIG. 13 Schematic Depicting the Interaction of Polypeptide Chains toForm a Tetrameric Diabody Molecule

NH₂ and COOH represent the amino-terminus and carboxy terminus,respectively of each polypeptide chain. S represents the at least onedisulfide bond between a cysteine residue in the second linker sequencethe Fc bearing, ‘heavier,’ polypeptide chain and a cysteine residue inthe C-terminal sequence of the non-Fc bearing, ‘lighter,’ polypeptidechain. VL and VH indicate the variable light domain and variable heavydomain, respectively. Dotted and dashed lines are to distinguish betweenpolypeptide chains and, in particular, represent the first linkerportions of said heavier chains or the linker of said lighter chains.CH2 and CH3 represent the CH2 and CH3 constant domains of an Fc domain.h2B6 Fv and h3G8 Fv indicate an epitope binding site specific for CD32Band CD16, respectively.

FIG. 14 Schematic Representation of Polypeptides Chains Containing FcDomains which Form Covalent Bispecific Diabodies

Representation of polypeptide constructs which form the diabodymolecules of the invention (each construct is described from the aminoterminus (“n”), left side of the construct, to the carboxy terminus(“c”), right side of figure). Construct (9) (SEQ ID NO:19) comprised n—aHinge/Fc domain of human IgG1—the VL domain Hu3G8—linker (GGGSGGGG (SEQID NO: 10))—the VH domain of Hu2B6—linker (GGGSGGGG (SEQ ID NO:10))—anda C-terminal LGGC sequence-c; construct (10) (SEQ ID NO:20) comprisedn—an Fc domain of human IgG1—the VL domain Hu3G8—linker (GGGSGGGG (SEQID NO:10))—the VH domain of Hu2B6—linker (GGGSGGGG (SEQ ID NO: 10))—anda C-terminal LGGC sequence-c; construct (11) (SEQ ID NO:21) comprisedn—the VL domain Hu2B6 (G105C)—linker (GGGSGGGG (SEQ ID NO:10))—the VHdomain of Hu3G8—and a C-terminal hinge/Fc domain of human IgG1 withamino acid substitution A215V-c; construct (12) (SEQ ID NO:22) comprisedn—the VL domain Hu3G8—linker (GGGSGGGG (SEQ ID NO: 10))—the VH domain ofHu2B6 (G44C)—and a C-terminal FNRGEC (SEQ ID NO:23) sequence-c.

FIG. 15A-B SDS-PAGE and Western Blot Analysis of Affinity TetramericDiabodies

Diabodies produced by recombinant expression systems cotransfected withvectors expressing constructs 10 and 1, constructs 9 and 1, andconstructs 11 and 12 were subjected to SDS-PAGE analysis non-reducingconditions (A) and Western Blot analysis using goat anti-human IgG1 H+Las the probe (B). Proteins in the SDS-PAGE gel were visualized withSimply Blue Safestain (Invitrogen). For both panels A and B, diabodymolecules comprising constructs 10 and 1, constructs 9 and 1, andconstructs 11 and 12A are in lanes 1, 2 and 3, respectively.

FIG. 16 Binding of Diabody Molecules Comprising Fc Domains andEngineered Interchain Disulfide Bonds to sCD32B and sCD16A

The binding of diabody molecules comprising Fc domains and engineereddisulfide bonds between the ‘lighter’ and ‘heavier’ polypeptide chainsto sCD32B and sCD16A was assayed in a sandwich ELISA. Diabodies assayedwere produced by 3 recombinant expression systems: expressing constructs1 and 10, expressing constructs 1 and 9, and expressing constructs 11and 12, respectively. sCD32B was used as the target protein. Thesecondary probe was HRP conjugated sCD16A. Binding of h3G8 was used ascontrol.

FIG. 17 Schematic Representation of Polyprotein Precursor of DiabodyMolecule and Shcematic Representation of Polypeptide Chains ContainingLambda Light Chain and/or Hinge Domains

Representation of polypeptide constructs which comprise the diabodymolecules of the invention (each construct is described from the aminoterminus (“n”), left side of the construct, to the carboxy terminus(“c”), right side of figure). Construct (13) (SEQ ID NO:97) comprised,n—VL domain 3G8—a first linker (GGGSGGGG (SEQ ID NO:10))—the VH domainof 2.4G2VH—a second linker (LGGC)-furin recognition site (RAKR (SEQ IDNO:95))—VL domain of 2.4G2—a third linker (GGGSGGG (SEQ ID NO:10)—VHdomain of 3G8—and a C-terminal LGGC domain; (nucleotide sequenceencoding SEQ ID NO:97 is provided in SEQ ID NO:98). Construct (14) (SEQID NO:99) comprised, n—VL domain 3G8—a first linker (GGGSGGGG (SEQ IDNO:10))—the VH domain of 2.4G2VH—a second linker (LGGC)-furinrecognition site (RAKR (SEQ ID NO:95))—FMD (Foot and Mouth Disease VirusProtease C3) site—VL domain of 2.4G2—a third linker (GGGSGGG (SEQ IDNO:10)—VH domain of 3G8—and a C-terminal LGGC domain; (nucleotidesequence encoding SEQ ID NO:99 is provided in SEQ ID NO:100). Construct(15) (SEQ ID NO:101) comprised, n—VL domain Hu2B6—a linker (GGGSGGGG(SEQ ID NO:10))—the VH domain of Hu3G8—and a C-terminal FNRGEC (SEQ IDNO:23) domain; (nucleotide sequence encoding SEQ ID NO:101 is providedin SEQ ID NO:102). Construct (16) (SEQ ID NO:103) comprised, n—VL domainHu3G8—a linker (GGGSGGGG (SEQ ID NO:10))—the VH domain of Hu2B6—and aC-terminal VEPKSC (SEQ ID NO:79) domain; (nucleotide sequence encodingSEQ ID NO:103 is provided in SEQ ID NO: 104).

FIG. 18 Binding of Diabody Molecules Derived from a PolyproteinPrecursor Molecule to mCD32B and sCD16A

The binding of diabody molecules derived from the polyprotein precursormolecule construct 13 (SEQ ID NO:97) to murine CD32B (mCD32B) andsoluble CD16A (sCD16A) was assayed in a sandwich ELISA. mCD32B was usedas the target protein. The secondary probe was biotin conjugated sCD16A.

FIG. 19 Binding of Diabody Molecules Comprising Lambda Chain and/orHinge Domains to sCD32B and sCD16A

The binding of diabody molecules comprising domains derived from theC-terminus of the human lambda light chain and/or the hinge domain ofIgG to sCD32B and sCD16A was assayed and compared to the diabodycomprising constructs 1 and 2 (FIG. 5) in a sandwich ELISA. Diabodiesassayed were produced by the recombinant expression system expressingconstructs 15 and 16 (SEQ ID NO:101 and SEQ ID NO:103, respectively).sCD32B was used as the target protein. The secondary probe was HRPconjugated sCD16A. Bars with small boxes represent the construct 15/16combination while bars with large boxes represent construct 1/2combination.

5. DESCRIPTION OF THE PREFERRED EMBODIMENTS

Each polypeptide chain of the diabody molecule comprises a VL domain anda VH domain, which are covalently linked such that the domains areconstrained from self assembly. Interaction of two of the polypeptidechains will produce two VL-VH pairings, forming two eptipoe bindingsites, i.e., a bivalent molecule. Neither the VH or VL domain isconstrained to any position within the polypeptide chain, i.e.,restricted to the amino (N) or carboxy (C) teminus, nor are the domainsrestricted in their relative positions to one another, i.e., the VLdomain may be N-terminal to the VH domain and vice-versa. The onlyrestriction is that a complimentary polypeptide chain be available inorder to form functional diabody. Where the VL and VH domains arederived from the same antibody, the two complimentary polypeptide chainsmay be identical. For example, where the binding domains are derivedfrom an antibody specific for epitope A (i.e., the binding domain isformed from a VL_(A)-VH_(A) interaction), each polypeptide will comprisea VH_(A) and a VL_(A). Homodimerization of two polypeptide chains of theantibody will result in the formation two VL_(A)-VH_(A) binding sites,resulting in a bivalent monospecific antibody. Where the VL and VHdomains are derived from antibodies specific for different antigens,formation of a functional bispecific diabody requires the interaction oftwo different polypeptide chains, i.e., formation of a heterodimer. Forexample, for a bispecific diabody, one polypeptide chain will comprise aVL_(A) and a VL_(B); homodimerization of said chain will result in theformation of two VL_(A)-VH_(B) binding sites, either of no binding or ofunpredictable binding. In contrast, where two differing polypeptidechains are free to interact, e.g., in a recombinant expression system,one comprising a VL_(A) and a VH_(B) and the other comprising a VL_(B)and a VH_(A), two differing binding sites will form: VL_(A)-VH_(A) andVL_(B)-VH_(B). For all diabody polypeptide chain pairs, the possibly ofmisalignment or mis-binding of the two chains is a possibility, i.e.,interaction of VL-VL or VH-VH domains; however, purification offunctional diabodies is easily managed based on the immunospecificity ofthe properly dimerized binding site using any affinity based methodknown in the are or exemplified herein, e.g., affinity chromatography.

In other embodiments, one or more of the polypeptide chains of thediabody comprises an Fc domain. Fc domains in the polypeptide chains ofthe diabody molecules preferentially dimerize, resulting in theformation of a diabody molecule that exhibits immunoglobulin-likeproperties, e.g., Fc-FcγR, interactions. Fc comprising diabodies may bedimers, e.g., comprised of two polypeptide chains, each comprising a VHdomain, a VL domain and an Fc domain. Dimerization of said polypeptidechains results in a bivalent diabody comprising an Fc domain, albeitwith a structure distinct from that of an unmodified bivalent antibody(FIG. 11). Such diabody molecules will exhibit altered phenotypesrelative to a wild-type immunoglobulin, e.g., altered serum half-life,binding properties, etc. In other embodiments, diabody moleculescomprising Fc domains may be tetramers. Such tertramers comprise two‘heavier’ polypeptide chains, i.e. a polypeptide chain comprising a VL,a VH and an Fc domain, and two ‘lighter’ polypeptide chains, i.e.,polypeptide chain comprising a VL and a VH. Said lighter and heavierchains interact to form a monomer, and said monomers interact via theirunpaired Fc domains to form an Ig-like molecule. Such an Ig-like diabodyis tetravalent and may be monospecific, bispecific or tetraspecific.

The at least two binding sites of the diabody molecule can recognize thesame or different epitopes. Different epitopes can be from the sameantigen or epitopes from different antigens. In one embodiment, theepitopes are from different cells. In another embodiment, the epitopesare cell surface antigens on the same cell or virus. The epitopesbinding sites can recognize any antigen to which an antibody can begenerated. For example, proteins, nucleic acids, bacterial toxins, cellsurface markers, autoimmune markers, viral proteins, drugs, etc. Inparticular aspects, at least one epitope binding site of the diabody isspecific for an antigen on a particular cell, such as a B-cell orT-cell, a phagocytotic cell, a natural killer (NK) cell or a dendriticcell.

Each domain of the polypeptide chain of the diabody, i.e., the VL, VHand FC domain may be separated by a peptide linker. The peptide linkermay be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. amino acids. In certainembodiments the amino acid linker sequence is GGGSGGGG (SEQ ID NO:10)encoded by the nucleic acid sequence (SEQ ID NO:76).

In certain embodiments, each polypeptide chain of the diabody moleculeis engineered to comprise at least one cysteine residue that willinteract with a counterpart at least one cysteine residue on a secondpolypeptide chain of the invention to form an inter-chain disulfidebond. Said interchain disulfide bonds serve to stabilize the diabodymolecule, improving expression and recovery in recombinant systems,resulting in a stable and consistent formulation as well as improvingthe stability of the isolated and/or purified product in vivo. Said atleast one cysteine residue may be introduced as a single amino acid oras part of larger amino-acid sequence, e.g hinge domain, in any portionof the polypeptide chain. In a specific embodiment, said at least onecysteine residue is engineered to occur at the C-terminus of thepolypeptide chain. In some embodiments, said at least one cysteineresidue in introduced into the polypeptide chain within the amino acidsequence LGGC. In a specific embodiment, the C-terminus of thepolypeptide chain comprising the diabody molecule of the inventioncomprises the amino acid sequence LGGC. In another embodiment, said atleast one cysteine residue is introduced into the polypeptide within anamino acid sequence comprising a hinge domain, e.g. SEQ ID NO:1 or SEQID NO:4. In a specific embodiment, the C-terminus of a polypeptide chainof the diabody molecule of the invention comprises the amino acidsequence of an IgG hinge domain, e.g. SEQ ID NO:1. In anotherembodiment, the C-terminus of a polypeptide chain of a diabody moleculeof the invention comprises the amino acid sequence VEPKSC (SEQ IDNO:79), which can be encoded by nucleotide sequence (SEQ ID NO:80). Inother embodiments, said at least one cysteine residue in introduced intothe polypeptide chain within the amino acid sequence LGGCFNRGEC (SEQ IDNO:17), which can be encoded by the nucleotide sequence (SEQ ID NO:78).In a specific embodiment, the C-terminus of a polypeptide chaincomprising the diabody of the invention comprises the amino acidsequence LGGCFNRGEC (SEQ ID NO:17), which can be encoded by thenucleotide sequence (SEQ ID NO:78). In yet other embodiments, said atleast one cysteine residue in introduced into the polypeptide chainwithin the amino acid sequence FNRGEC (SEQ ID NO:23), which can beencoded by the nucleotide sequence (SEQ ID NO:77). In a specificembodiment, the C-terminus of a polypeptide chain comprising the diabodyof the invention comprises the amino acid sequence FNRGEC (SEQ IDNO:23), which can be encoded by the nucleotide sequence (SEQ ID NO:77).

In certain embodiments, the diabody molecule comprises at least twopolypeptide chains, each of which comprise the amino acid sequence LGGCand are covalently linked by a disulfide bond between the cysteineresidues in said LGGC sequences. In another specific embodiment, thediabody molecule comprises at least two polypeptide chains, one of whichcomprises the sequence FNRGEC (SEQ ID NO:23) while the other comprises ahinge domain (containing at least one cysteine residue), wherein said atleast two polypeptide chains are covalently linked by a disulfide bondbetween the cysteine residue in FNRGEC (SEQ ID NO:23) and a cysteineresidue in the hinge domain. In particular aspects, the cysteine residueresponsible for the disulfide bond located in the hinge domain isCys-128 (as numbered according to Kabat EU; located in the hinge domainof an unmodified, intact IgG heavy chain) and the counterpart cysteineresidue in SEQ ID NO:23 is Cys-214 (as numbered according to Kabat EU;located at the C-terminus of an unmodified, intact IgG light chain)(Elkabetz et al., 2005, J. Biol. Chem. 280:14402-14412; herebyincorporated by reference herein in its entirety). In yet otherembodiments, the at least one cysteine residue is engineered to occur atthe N-terminus of the amino acid chain. In still other embodiments, theat least one cysteine residue is engineered to occur in the linkerportion of the polypeptide chain of the diabody molecule. In furtherembodiments, the VH or VL domain is engineered to comprise at least oneamino acid modification relative to the parental VH or VL domain suchthat said amino acid modification comprises a substitution of a parentalamino acid with cysteine.

The invention encompasses diabody molecules comprising an Fc domain orportion thereof (e.g. a CH2 domain, or CH3 domain). The Fc domain orportion thereof may be derived from any immunoglobulin isotype orallotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. Inpreferred embodiments, the Fc domain (or portion thereof) is derivedfrom IgG. In specific embodiments, the IgG isotype is IgG1, IgG2, IgG3or IgG4 or an allotype thereof. In one embodiment, the diabody moleculecomprises an Fc domain, which Fc domain comprises a CH2 domain and CH3domain independently selected from any immunoglobulin isotype (i.e. anFc domain comprising the CH2 domain derived from IgG and the CH3 domainderived form IgE, or the CH2 domain derived from IgG1 and the CH3 domainderived from IgG2, etc.). Said Fc domain may be engineered into apolypeptide chain comprising the diabody molecule of the invention inany position relative to other domains or portions of said polypeptidechain (e.g., the Fc domain, or portion thereof, may be c-terminal toboth the VL and VH domains of the polypeptide of the chain; may ben-terminal to both the VL and VH domains; or may be N-terminal to onedomain and c-terminal to another (i.e., between two domains of thepolypeptide chain)).

The present invention also encompasses molecules comprising a hingedomain. The hinge domain be derived from any immunoglobulin isotype orallotype including IgA, IgD, IgG, IgE and IgM. In preferred embodiments,the hinge domain is derived from IgG, wherein the IgG isotype is IgG1,IgG2, IgG3 or IgG4, or an allotype thereof. Said hinge domain may beengineered into a polypeptide chain comprising the diabody moleculetogether with an Fc domain such that the diabody molecule comprises ahinge-Fc domain. In certain embodiments, the hinge and Fc domain areindependently selected from any immunoglobulin isotype known in the artor exemplified herein. In other embodiments the hinge and Fc domain areseparated by at least one other domain of the polypeptide chain, e.g.,the VL domain. The hinge domain, or optionally the hinge-Fc domain, maybe engineered in to a polypeptide of the invention in any positionrelative to other domains or portions of said polypeptide chain. Incertain embodiments, a polypeptide chain of the invention comprises ahinge domain, which hinge domain is at the C-terminus of the polypeptidechain, wherein said polypeptide chain does not comprise an Fc domain. Inyet other embodiments, a polypeptide chain of the invention comprises ahinge-Fc domain, which hinge-Fc domain is at the C-terminus of thepolypeptide chain. In further embodiments, a polypeptide chain of theinvention comprises a hinge-Fc domain, which hinge-Fc domain is at theN-terminus of the polypeptide chain.

As discussed above, the invention encompasses multimers of polypeptidechains, each of which polypeptide chains comprise a VH and VL domain. Incertain aspects, the polypeptide chains in said multimers furthercomprise an Fc domain. Dimerization of the Fc domains leads to formationof a diabody molecule that exhibits immunoglobulin-like functionality,i.e., Fc mediated function (e.g., Fc-FcγR interaction, complementbinding, etc.). In certain embodiments, the VL and VH domains comprisingeach polypeptide chain have the same specificity, and said diabodymolecule is bivalent and monospecific. In other embodiments, the VL andVH domains comprising each polypeptide chain have differing specificityand the diabody is bivalent and bispecific.

In yet other embodiments, diabody molecules of the invention encompasstetramers of polypeptide chains, each of which polypeptide chaincomprises a VH and VL domain. In certain embodiments, two polypeptidechains of the tetramer further comprise an Fc domain. The tetramer istherefore comprised of two ‘heavier’ polypeptide chains, each comprisinga VL, VH and Fc domain, and two ‘lighter’ polypeptide chains, comprisinga VL and VH domain. Interaction of a heavier and lighter chain into abivalent monomer coupled with dimerization of said monomers via the Fcdomains of the heavier chains will lead to formation of a tetravalentimmunoglobulin-like molecule (exemplified in Example 6.2 and Example6.3). In certain aspects the monomers are the same, and the tetravalentdiabody molecule is monospecific or bispecific. In other aspects themonomers are different, and the tetra valent molecule is bispecific ortetraspecific.

Formation of a tetraspecific diabody molecule as described suprarequires the interaction of four differing polypeptide chains. Suchinteractions are difficult to achieve with efficiency within a singlecell recombinant production system, due to the many variants ofpotential chain mispairings. One solution to increase the probability ofmispairings, is to engineer “knobs-into-holes” type mutations into thedesired polypeptide chain pairs. Such mutations favor heterodimerizationover homodimerization. For example, with respect to Fc-Fc-interactions,an amino acid substitution (preferably a substitution with an amino acidcomprising a bulky side group forming a ‘knob’, e.g., tryptophan) can beintroduced into the CH2 or CH3 domain such that steric interference willprevent interaction with a similarly mutated domain and will obligatethe mutated domain to pair with a domain into which a complementary, oraccommodating mutation has been engineered, i.e., ‘the hole’ (e.g., asubstitution with glycine). Such sets of mutations can be engineeredinto any pair of polypeptides comprising the diabody molecule, andfurther, engineered into any portion of the polypeptides chains of saidpair. Methods of protein engineering to favor heterodimerization overhomodimerization are well known in the art, in particular with respectto the engineering of immunoglobulin-like molecules, and are encompassedherein (see e.g., Ridgway et al., 1996, Protein Engr. 9:617-621, Atwellet al., 1997, J. Mol. Biol. 270: 26-35, and Xie et al., 2005, J.Immunol. Methods 296:95-101; each of which is hereby incorporated hereinby reference in its entirety).

The invention also encompasses diabody molecules comprising variant Fcor variant hinge-Fc domains (or portion thereof), which variant Fcdomain comprises at least one amino acid modification (e.g.substitution, insertion deletion) relative to a comparable wild-type Fcdomain or hinge-Fc domain (or portion thereof). Molecules comprisingvariant Fc domains or hinge-Fc domains (or portion thereof) (e.g.,antibodies) normally have altered phenotypes relative to moleculescomprising wild-type Fc domains or hinge-Fc domains or portions thereof.The variant phenotype may be expressed as altered serum half-life,altered stability, altered susceptibility to cellular enzymes or alteredeffector function as assayed in an NK dependent or macrophage dependentassay. Fc domain variants identified as altering effector function aredisclosed in International Application WO04/063351, U.S. PatentApplication Publications 2005/0037000 and 2005/0064514, U.S. ProvisionalApplications 60/626,510, filed Nov. 10, 2004, 60/636,663, filed Dec. 15,2004, and 60/781,564, filed Mar. 10, 2006, and U.S. patent applicationsSer. No. 11/271,140, filed Nov. 10, 2005, and Ser. No. 11/305,787, filedDec. 15, 2005, concurrent applications of the Inventors, each of whichis incorporated by reference in its entirety.

The bispecific diabodies of the invention can simultaneously bind twoseparate and distinct epitopes. In certain embodiments the epitopes arefrom the same antigen. In other embodiments, the epitopes are fromdifferent antigens. In preferred embodiments, at least one epitopebinding site is specific for a determinant expressed on an immuneeffector cell (e.g. CD3, CD16, CD32, CD64, etc.) which are expressed onT lymphocytes, natural killer (NK) cells or other mononuclear cells. Inone embodiment, the diabody molecule binds to the effector celldeterminant and also activates said effector cell. In this regard,diabody molecules of the invention may exhibit Ig-like functionalityindependent of whether they further comprise an Fc domain (e.g., asassayed in any effector function assay known in the art or exemplifiedherein (e.g., ADCC assay). In certain embodiments the bispecific diabodyof the invention binds both a cancer antigen on a tumor cell and aneffector cell determinant while activating said cell. In alternativeembodiments, the bispecific diabody or diabody molecule of the inventionmay inhibit activation of a target, e.g., effector, cell bysimultaneously binding, and thus linking, an activating and inhibitoryreceptor on the same cell (e.g., bind both CD32A and CD32B, BCR andCD32B, or IgERI and CD32B) as described supra (see, Background Section).In a further aspect of this embodiment, the bispecific diabody mayexhibit anti-viral properties by simultaneously binding two neutralizingepitopes on a virus (e.g., RSV epitopes; WNV epitopes such as E16 andE53).

In certain embodiments, bispecific diabody molecules of the inventionoffer unique opportunities to target specific cell types. For example,the bispecific diabody or diabody molecule can be engineered to comprisea combination of epitope binding sites that recognize a set of antigensunique to a target cell or tissue type. Additionally, where either orboth of the individual antigens is/are fairly common separately in othertissue and/or cell types, low affinity biding domains can be used toconstruct the diabody or diabody molecule. Such low affinity bindingdomains will be unable to bind to the individual epitope or antigen withsufficient avidity for therapeutic purposes. However, where bothepitopes or antigens are present on a single target cell or tissue, theavidity of the diabody or diabody molecule for the cell or tissue,relative to a cell or tissue expressing only one of the antigens, willbe increased such that said cell or tissue can be effectively targetedby the invention. Such a bispecific molecule can exhibit enhancedbinding to one or both of its target antigens on cells expressing bothof said antigens relative to a monospecific diabody or an antibody witha specificity to only one of the antigens.

Preferably, the binding properties of the diabodies of the invention arecharacterized by in vitro functional assays for determining bindingactivity and/or one or more FcγR mediator effector cell functions(mediated via Fc-FcγR interactions or by the immunospecific binding of adiabody molecule to an FcγR) (See Section 5.4.2 and 5.4.3). Theaffinities and binding properties of the molecules, e.g., diabodies, ofthe invention for an FcγR can be determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining binding domain-antigen or Fc-FcγR interactions, i.e.,specific binding of an antigen to a binding domain or specific bindingof an Fc region to an FcγR, respectively, including but not limited toELISA assay, surface plasmon resonance assay, immunoprecipitation assays(See Section 5.4.2). In most preferred embodiments, the molecules of theinvention have similar binding properties in in vivo models (such asthose described and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude molecules of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo.

In some embodiments, molecules of the invention are engineered tocomprise an altered glycosylation pattern or an altered glycoformrelative to the comparable portion of the template molecule. Engineeredglycoforms may be useful for a variety of purposes, including, but notlimited to, enhancing effector function. Engineered glycoforms may begenerated by any method known to one skilled in the art, for example byusing engineered or variant expression strains, by co-expression withone or more enzymes, for example, DI N-acetylglucosaminyltransferase III(GnTI11), by expressing a diabody of the invention in various organismsor cell lines from various organisms, or by modifying carbohydrate(s)after the diabody has been expressed and purified. Methods forgenerating engineered glycoforms are known in the art, and include butare not limited to those described in Umana et al, 1999, Nat. Biotechnol17:176-180; Davies et al., 2001 Biotechnol Bioeng 74:288-294; Shields etal, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J BiolChem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370;U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc.Princeton, N.J.); GlycoMAb™ glycosylation engineering technology(GLYCART biotechnology AG, Zurich, Switzerland); each of which isincorporated herein by reference in its entirety. See, e.g., WO00061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336:1239-49 each of which is incorporated herein by reference in itsentirety.

The invention further encompasses incorporation of unnatural amino acidsto generate the diabodies of the invention. Such methods are known tothose skilled in the art such as those using the natural biosyntheticmachinery to allow incorporation of unnatural amino acids into proteins,see, e.g., Wang et al., 2002 Chem. Comm. 1: 1-11; Wang et al., 2001,Science, 292: 498-500; van Hest et al., 2001. Chem. Comm. 19: 1897-1904,each of which is incorporated herein by reference in its entirety.Alternative strategies focus on the enzymes responsible for thebiosynthesis of amino acyl-tRNA, see, e.g., Tang et al., 2001, J. Am.Chem. 123(44): 11089-11090; Kiick et al., 2001, FEBS Lett. 505(3): 465;each of which is incorporated herein by reference in its entirety.

In some embodiments, the invention encompasses methods of modifying aVL,VH or Fc domain of a molecule of the invention by adding or deletinga glycosylation site. Methods for modifying the carbohydrate of proteinsare well known in the art and encompassed within the invention, see,e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S. Publication No. US2002/0028486; WO 03/035835; U.S. Publication No. 2003/0115614; U.S. Pat.No. 6,218,149; U.S. Pat. No. 6,472,511; all of which are incorporatedherein by reference in their entirety.

5.1 Diabody Binding Domains

The diabodies of the present invention comprise antigen binding domainsgenerally derived from immunoglobulins or antibodies. The antibodiesfrom which the binding domains used in the methods of the invention arederived may be from any animal origin including birds and mammals (e.g.,human, non-human primate, murine, donkey, sheep, rabbit, goat, guineapig, camel, horse, or chicken). Preferably, the antibodies are human orhumanized monoclonal antibodies. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or libraries of synthetic human immunoglobulin codingsequences or from mice that express antibodies from human genes.

The invention contemplates the use of any antibodies known in the artfor the treatment and/or prevention of cancer, autoimmune disease,inflammatory disease or infectious disease as source of binding domainsfor the diabodies of the invention. Non-limiting examples of knowncancer antibodies are provided in section 5.7.1 as well as otherantibodies specific for the listed target antigens and antibodiesagainst the cancer antigens listed in section 5.6.1; nonlimitingexamples of known antibodies for the treatment and/or prevention ofautoimmune disease and inflammatory disease are provided in section5.7.2. as well as antibodies against the listed target antigens andantibodies against the antigens listed in section 5.6.2; in otherembodiments antibodies against epitopes associated with infectiousdiseases as listed in Section 5.6.3 can be used. In certain embodiments,the antibodies comprise a variant Fc region comprising one or more aminoacid modifications, which have been identified by the methods of theinvention to have a conferred effector function and/or enhanced affinityfor FcγRIIB and a decreased affinity for FcγRIIIA relative to acomparable molecule comprising a wild type Fc region. A non-limitingexample of the antibodies that are used for the treatment or preventionof inflammatory disorders which can be engineered according to theinvention is presented in Table 9, and a non-limiting example of theantibodies that are used for the treatment or prevention of autoimmunedisorder is presented in Table 10.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use diabodies withvariable domains derived from human, chimeric or humanized antibodies.Variable domains from completely human antibodies are particularlydesirable for therapeutic treatment of human subjects. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

A humanized antibody is an antibody, a variant or a fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodymay comprise substantially all of at least one, and typically two,variable domains in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin (i.e., donor antibody)and all or substantially all of the framework regions are those of ahuman immunoglobulin consensus sequence.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the donorantibody. Such mutations, however, are preferably not extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental framework region (FR) and CDR sequences, moreoften 90%, and most preferably greater than 95%. Humanized antibodiescan be produced using variety of techniques known in the art, includingbut not limited to, CDR-grafting (European Patent No. EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (European PatentNos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology28(4/5):489-498; Studnicka et al., 1994, Protein Engineering7(6):805-814; and Roguska et al., 1994, Proc Natl Acad Sci USA91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and techniquesdisclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089,International Publication No. WO 9317105, Tan et al., 2002, J. Immunol.169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al.,2000, Methods 20:267-79, Baca et al., 1997, J. Biol. Chem. 272:10678-84,Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, CancerRes. 55 (23 Supp):5973s-5977s, Couto et al., 1995, Cancer Res.55:1717-22, Sandhu, 1994, Gene 150:409-10, Pedersen et al., 1994, J.Mol. Biol. 235:959-73, Jones et al., 1986, Nature 321:522-525, Riechmannet al., 1988, Nature 332:323, and Presta, 1992, Curr. Op. Struct. Biol.2:593-596. Often, framework residues in the framework regions will besubstituted with the corresponding residue from the CDR donor antibodyto alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; U.S.Publication Nos. 2004/0049014 and 2003/0229208; U.S. Pat. Nos.6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 andRiechmann et al., 1988, Nature 332:323, all of which are incorporatedherein by reference in their entireties.).

In a most preferred embodiment, the humanized binding domainspecifically binds to the same epitope as the donor murine antibody. Itwill be appreciated by one skilled in the art that the inventionencompasses CDR grafting of antibodies in general. Thus, the donor andacceptor antibodies may be derived from animals of the same species andeven same antibody class or sub-class. More usually, however, the donorand acceptor antibodies are derived from animals of different species.Typically the donor antibody is a non-human antibody, such as a rodentMAb, and the acceptor antibody is a human antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

The diabodies used in the methods of the invention include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the diabody. For example, but not by way of limitation, thediabody derivatives include diabodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 6,311,415,5,807,715, 4,816,567, and 4,816,397, which are incorporated herein byreference in their entirety.

Often, framework residues in the framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323,which are incorporated herein by reference in their entireties.)

Monoclonal antibodies from which binding domains of the diabodies of theinvention can be prepared using a wide variety of techniques known inthe art including the use of hybridoma, recombinant, and phage displaytechnologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas, pp. 563-681 (Elsevier, N.Y., 1981) (both of which areincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones. Antigens of interest include, but are not limited to, antigensassociated with the cancers provided in section 5.8.1, antigensassociated with the autoimmune diseases and inflammatory diseasesprovided in section 5.8.2, antigens associated with the infectiousdiseases provided in section 5.8.3, and the toxins provided in section5.8.4.

Antibodies can also be generated using various phage display methodsknown in the art. In phage display methods, functional antibody domainsare displayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In a particular embodiment, suchphage can be utilized to display antigen binding domains, such as Faband Fv or disulfide-bond stabilized Fv, expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage, including fd and M13. Theantigen binding domains are expressed as a recombinantly fused proteinto either the phage gene III or gene VIII protein. Examples of phagedisplay methods that can be used to make the immunoglobulins, orfragments thereof, of the present invention include those disclosed inBrinkman et aL., J. Immunol. Methods, 182:41-50, 1995; Ames et al., J.Immunol. Methods, 184:177-186, 1995; Kettleborough et al., Eur. J.Immunol., 24:952-958, 1994; Persic et al., Gene, 187:9-18, 1997; Burtonet al., Advances in Immunology, 57:191-280, 1994; PCT Application No.PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Phage display technology can be used to increase the affinity of anantibody for its antigen. This technique would be useful in obtaininghigh affinity antibodies. The technology, referred to as affinitymaturation, employs mutagenesis or CDR walking and re-selection usingthe cognate antigen to identify antibodies that bind with higheraffinity to the antigen when compared with the initial or parentalantibody (See, e.g., Glaser et al., 1992, J. Immunology 149:3903).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased binding affinity for the antigen can bescreened by contacting the immobilized mutants with labeled antigen. Anyscreening method known in the art can be used to identify mutantantibodies with increased avidity to the antigen (e.g., ELISA) (See Wuet al., 1998, Proc Natl. Acad Sci. USA 95:6037; Yelton et al., 1995, J.Immunology 155:1994). CDR walking which randomizes the light chain isalso possible (See Schier et al., 1996, J. Mol. Bio. 263:551).

The present invention also encompasses the use of binding domainscomprising the amino acid sequence of any of the binding domainsdescribed herein or known in the art with mutations (e.g., one or moreamino acid substitutions) in the framework or CDR regions. Preferably,mutations in these binding domains maintain or enhance the avidityand/or affinity of the binding domains for FcγRIIB to which theyimmunospecifically bind. Standard techniques known to those skilled inthe art (e.g., immunoassays) can be used to assay the affinity of anantibody for a particular antigen.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

5.1.1 Diabodies Comprising Eptiope Binding Sites whichImmunospecifically Bind FcγRIIB

In a particular embodiment, at least one of the binding domains of thediabodies of the invention agonizes at least one activity of FcγRIIB. Inone embodiment of the invention, said activity is inhibition of B cellreceptor-mediated signaling. In another embodiment, the binding domaininhibits activation of B cells, B cell proliferation, antibodyproduction, intracellular calcium influx of B cells, cell cycleprogression, or activity of one or more downstream signaling moleculesin the FcγRIIB signal transduction pathway. In yet another embodiment,the binding domain enhances phosphorylation of FcγRIIB or SHIPrecruitment. In a further embodiment of the invention, the bindingdomain inhibits MAP kinase activity or Akt recruitment in the B cellreceptor-mediated signaling pathway. In another embodiment, the bindingdomain agonizes FcγRIIB-mediated inhibition of FcεRI signaling. In aparticular embodiment, said binding domain inhibits FcεRI-induced mastcell activation, calcium mobilization, degranulation, cytokineproduction, or serotonin release. In another embodiment, the bindingdomains of the invention stimulate phosphorylation of FcγRIIB, stimulaterecruitment of SHIP, stimulate SHIP phosphorylation and its associationwith Shc, or inhibit activation of MAP kinase family members (e.g.,Erk1, Erk2, JNK, p38, etc.). In yet another embodiment, the bindingdomains of the invention enhance tyrosine phosphorylation of p62dok andits association with SHIP and rasGAP. In another embodiment, the bindingdomains of the invention inhibit FcγR-mediated phagocytosis in monocytesor macrophages.

In another embodiment, the binding domains antagonize at least oneactivity of FcγRIIB. In one embodiment, said activity is activation of Bcell receptor-mediated signaling. In a particular embodiment, thebinding domains enhance B cell activity, B cell proliferation, antibodyproduction, intracellular calcium influx, or activity of one or moredownstream signaling molecules in the FcγRIIB signal transductionpathway. In yet another particular embodiment, the binding domainsdecrease phosphorylation of FcγRIIB or SHIP recruitment. In a furtherembodiment of the invention, the binding domains enhance MAP kinaseactivity or Akt recruitment in the B cell receptor mediated signalingpathway. In another embodiment, the binding domains antagonizeFcγRIIB-mediated inhibition of FcεRI signaling. In a particularembodiment, the binding domains enhance FcεRI-induced mast cellactivation, calcium mobilization, degranulation, cytokine production, orserotonin release. In another embodiment, the binding domains inhibitphosphorylation of FcγRIIB, inhibit recruitment of SHIP, inhibit SHIPphosphorylation and its association with Shc, enhance activation of MAPkinase family members (e.g., Erk1, Erk2, JNK, p38, etc.). In yet anotherembodiment, the binding domains inhibit tyrosine phosphorylation ofp62dok and its association with SHIP and rasGAP. In another embodiment,the binding domains enhance FcγR-mediated phagocytosis in monocytes ormacrophages. In another embodiment, the binding domains preventphagocytosis, clearance of opsonized particles by splenic macrophages.

In other embodiments, at least one of the binding domains can be used totarget the diabodies of the invention to cells that express FcγRIIB.

In one particular embodiment, one of the binding domains is derived froma mouse monoclonal antibody produced by clone 2B6 or 3H7, having ATCCaccession numbers PTA-4591 and PTA-4592, respectively. Hybridomasproducing antibodies 2B6 and 3H7 have been deposited with the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on Aug. 13, 2002 under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedures, and assigned accession numbersPTA-4591 (for hybridoma producing 2B6) and PTA-4592 (for hybridomaproducing 3H7), respectively, and are incorporated herein by reference.In a preferred embodiment, the binding domains are human or have beenhumanized, preferably are derived from a humanized version of theantibody produced by clone 3H7 or 2B6.

The invention also encompasses diabodies with binding domains from otherantibodies, that specifically bind FcγRIIB, preferably human FcγRIIB,more preferably native human FcγRIIB, that are derived from clonesincluding but not limited to 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCCAccession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively. Hybridomas producing the above-identified clones weredeposited under the provisions of the Budapest Treaty with the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on May 7, 2004, and are incorporated herein by reference. Inpreferred embodiments, the binding domains from the antibodies describedabove are humanized.

In a specific embodiment, the binding domains used in the diabodies ofthe present invention are from an antibody or an antigen-bindingfragment thereof (e.g., comprising one or more complementarilydetermining regions (CDRs), preferably all 6 CDRs) of the antibodyproduced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In anotherembodiment, the binding domain binds to the same epitope as the mousemonoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2, respectively and/or competes with the mouse monoclonal antibodyproduced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 as determined,e.g., in an ELISA assay or other appropriate competitive immunoassay,and also binds FcγRIIB with a greater affinity than the binding domainbinds FcγRIIA.

The present invention also encompasses diabodies with binding domainscomprising an amino acid sequence of a variable heavy chain and/orvariable light chain that is at least 45%, at least 50%, at least 55%,at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or at least 99% identical to theamino acid sequence of the variable heavy chain and/or light chain ofthe mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2. The present invention further encompasses diabodies withbinding domains that specifically bind FcγRIIB with greater affinitythan said antibody or fragment thereof binds FcγRIIA, and that comprisean amino acid sequence of one or more CDRs that is at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% identical to the amino acid sequence of one or more CDRs ofthe mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2. The determination of percent identity of two amino acidsequences can be determined by any method known to one skilled in theart, including BLAST protein searches.

The present invention also encompasses the use of diabodies containingbinding domains that specifically bind FcγRIIB with greater affinitythan binding domain binds FcγRIIA, which are encoded by a nucleotidesequence that hybridizes to the nucleotide sequence of the mousemonoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or1F2 under stringent conditions. In a preferred embodiment, the bindingdomain specifically binds FcγRIIB with greater affinity than FcγRIIA,and comprises a variable light chain and/or variable heavy chain encodedby a nucleotide sequence that hybridizes under stringent conditions tothe nucleotide sequence of the variable light chain and/or variableheavy chain of the mouse monoclonal antibody produced by clone 2B6, 3H7,1D5, 2E1, 2H9, 2D11, or 1F2 under stringent conditions. In anotherpreferred embodiment, the binding domains specifically bind FcγRIIB withgreater affinity than FcγRIIA, and comprise one or more CDRs encoded bya nucleotide sequence that hybridizes under stringent conditions to thenucleotide sequence of one or more CDRs of the mouse monoclonal antibodyproduced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. Stringenthybridization conditions include, but are not limited to, hybridizationto filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about45° C. followed by one or more washes in 0.2X SSC/0.1% SDS at about50-65° C., highly stringent conditions such as hybridization tofilter-bound DNA in 6×SSC at about 45° C. followed by one or more washesin 0.1X SSC/0.2% SDS at about 60° C., or any other stringenthybridization conditions known to those skilled in the art (see, forexample, Ausubel, F. M. et al., eds. 1989 Current Protocols in MolecularBiology, vol. 1, Green Publishing Associates, Inc. and John Wiley andSons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3, incorporated hereinby reference).

The present invention also encompasses the use of binding domainscomprising the amino acid sequence of any of the binding domainsdescribed above with mutations (e.g., one or more amino acidsubstitutions) in the framework or CDR regions. Preferably, mutations inthese binding domains maintain or enhance the avidity and/or affinity ofthe binding domains for FcγRIIB to which they immunospecifically bind.Standard techniques known to those skilled in the art (e.g.,immunoassays) can be used to assay the affinity of an antibody for aparticular antigen.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

In preferred embodiments, the binding domains are derived from humanizedantibodies. A humanized FcγRIIB specific antibody may comprisesubstantially all of at least one, and typically two, variable domainsin which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence.

The diabodies of present invention comprise humanized variable domainsspecific for FcγRIIB in which one or more regions of one or more CDRs ofthe heavy and/or light chain variable regions of a human antibody (therecipient antibody) have been substituted by analogous parts of one ormore CDRs of a donor monoclonal antibody which specifically bindsFcγRIIB, with a greater affinity than FcγRIIA, e.g., a monoclonalantibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. Inother embodiments, the humanized antibodies bind to the same epitope as2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, respectively.

In a preferred embodiment, the CDR regions of the humanized FcγRIIBbinding domain are derived from a murine antibody specific for FcγRIIB.In some embodiments, the humanized antibodies described herein comprisealterations, including but not limited to amino acid deletions,insertions, modifications, of the acceptor antibody, i.e., human, heavyand/or light chain variable domain framework regions that are necessaryfor retaining binding specificity of the donor monoclonal antibody. Insome embodiments, the framework regions of the humanized antibodiesdescribed herein does not necessarily consist of the precise amino acidsequence of the framework region of a natural occurring human antibodyvariable region, but contains various alterations, including but notlimited to amino acid deletions, insertions, modifications that alterthe property of the humanized antibody, for example, improve the bindingproperties of a humanized antibody region that is specific for the sametarget as the murine FcγRIIB specific antibody. In most preferredembodiments, a minimal number of alterations are made to the frameworkregion in order to avoid large-scale introductions of non-humanframework residues and to ensure minimal immunogenicity of the humanizedantibody in humans. The donor monoclonal antibody is preferably amonoclonal antibody produced by clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or1F2.

In a specific embodiment, the binding domain encompasses variabledomains of a CDR-grafted antibody which specifically binds FcγRIIB witha greater affinity than said antibody binds FcγRIIA, wherein theCDR-grafted antibody comprises a heavy chain variable region domaincomprising framework residues of the recipient antibody and residuesfrom the donor monoclonal antibody, which specifically binds FcγRIIBwith a greater affinity than said antibody binds FcγRIIA, e.g.,monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2. In another specific embodiment, the diabodies of the inventioncomprise variable domains from a CDR-grafted antibody which specificallybinds FcγRIIB with a greater affinity than said antibody binds FcγRIIA,wherein the CDR-grafted antibody comprises a light chain variable regiondomain comprising framework residues of the recipient antibody andresidues from the donor monoclonal antibody, which specifically bindsFc₇RIIB with a greater affinity than said antibody binds FcγRIIA, e.g.,monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2.

The humanized anti-FcγRIIB variable domains used in the invention mayhave a heavy chain variable region comprising the amino acid sequence ofCDR1 (SEQ ID NO:24 or SEQ ID NO:25) and/or CDR2 (SEQ ID NO:26 or SEQ IDNO:27) and/or CDR3 (SEQ ID NO:28 or SEQ ID NO:29) and/or a light chainvariable region comprising the amino acid sequence of CDR1 (SEQ ID NO:32or SEQ ID NO:33) and/or a CDR2 (SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, or SEQ ID NO:37) and/or CDR3 (SEQ ID NO:38 or SEQ ID NO:39).

In one specific embodiment, the diabody comprises variable domains froma humanized 2B6 antibody, wherein the VH region consists of the FRsegments from the human germline VH segment VH1-18 (Matsuda et al.,1998, J. Exp. Med. 188:2151062) and JH6 (Ravetch et al., 1981, Cell 27(3Pt. 2): 583-91), and one or more CDR regions of the 2B6 VH, having theamino acid sequence of SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28. Inone embodiment, the 2B6 VH has the amino acid sequence of SEQ ID NO:40.In another embodiment the 2B6 VH domain has the amino acid sequence ofHu2B6VH, SEQ ID NO:87, and can be encoded by the nucleotide sequence ofSEQ ID NO:88. In another specific embodiment, the diabody furthercomprises a VL region, which consists of the FR segments of the humangermline VL segment VK-A26 (Lautner-Rieske et al., 1992, Eur. J.Immunol. 22:1023-1029) and JK4 (Hieter et al., 1982, J. Biol. Chem.257:1516-22), and one or more CDR regions of 2B6VL, having the aminoacid sequence of SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,and SEQ ID NO:38. In one embodiment, the 2B6 VL has the amino acidsequence of SEQ ID NO:41; SEQ ID NO:42, or SEQ ID NO:43. In a specificembodiment, the 2B6 VL has the amino acid sequence of Hu2B6VL, SEQ IDNO:89, and can be encoded by the nucleotide sequence provided in SEQ IDNO:90.

In another specific embodiment, the diabody has variable domains from ahumanized 3H7 antibody, wherein the VH region consists of the FRsegments from a human germline VH segment and the CDR regions of the 3H7VH, having the amino acid sequence of SEQ ID NO. 37. In another specificembodiment, the humanized 3H7 antibody further comprises a VL regions,which consists of the FR segments of a human germline VL segment and theCDR regions of 3H7VL, having the amino acid sequence of SEQ ID NO:44.

In particular, binding domains immunospecifically bind to extracellulardomains of native human FcγRIIB, and comprise (or alternatively, consistof) CDR sequences of 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, in any ofthe following combinations: a VH CDR1 and a VL CDR1; a VH CDR1 and a VLCDR2; a VH CDR1 and a VL CDR3; a VH CDR2 and a VL CDR1; VH CDR2 and VLCDR2; a VH CDR2 and a VL CDR3; a VH CDR3 and a VH CDR1; a VH CDR3 and aVL CDR2; a VH CDR3 and a VL CDR3; a VH1 CDR1, a VH CDR2 and a VL CDR1; aVH CDR1, a VH CDR2 and a VL CDR2; a VH CDR1, a VH CDR2 and a VL CDR3; aVH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; aVH CDR2, a VH CDR2 and a VL CDR3; a VH CDR1, a VL CDR1 and a VL CDR2; aVH CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, a VL CDR1 and a VL CDR2; aVH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, a VL CDR1 and a VL CDR2; aVH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VH CDR3 and aVL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR1, a VHCDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1 and a VLCDR2; a VH CDR1, a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR3, a VL CDR1 and a VLCDR3; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR2 and a VLCDR3; a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VHCDR2, a VL CDR1, a VL CDR2, and a VL CDR3; a VH CDR1, a VH CDR3, a VLCDR1, a VL CDR2, and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1, a VLCDR2, and a VL CDR3; or any combination thereof of the VH CDRs and VLCDRs disclosed herein.

Antibodies for deriving binding domains to be included in the diabodiesof the invention may be further characterized by epitope mapping, sothat antibodies may be selected that have the greatest specificity forFcγRIIB compared to FcγRIIA. Epitope mapping methods of antibodies arewell known in the art and encompassed within the methods of theinvention. In certain embodiments fusion proteins comprising one or moreregions of FcγRIIB may be used in mapping the epitope of an antibody ofthe invention. In a specific embodiment, the fusion protein contains theamino acid sequence of a region of an FcγRIIB fused to the Fc portion ofhuman IgG2. Each fusion protein may further comprise amino acidsubstitutions and/or replacements of certain regions of the receptorwith the corresponding region from a homolog receptor, e.g., FcγRIIA, asshown in Table 2 below. pMGX125 and pMGX132 contain the IgG binding siteof the FcγRIIB receptor, the former with the C-terminus of FcγRIIB andthe latter with the C-terminus of FcγRIIA and can be used todifferentiate C-terminus binding. The others have FcγRIIA substitutionsin the IgG binding site and either the FcγIIA or FcγIIB N-terminus.These molecules can help determine the part of the receptor moleculewhere the antibodies bind. TABLE 2 List of the fusion proteins that maybe used to investigate the epitope of the monoclonal anti-FcγRIIBantibodies. Residues 172 to 180 belong to the IgG binding site ofFcγRIIA and B. The specific amino acids from FcγRIIA sequence are inbold. Plasmid Receptor N-terminus 172-180 SEQ ID NO: C-terminus pMGX125RIIb IIb KKFSRSDPN 45 APS------SS (IIb) pMGX126 RIIa/b IIa QKFSRLDPN 46APS------SS (IIb) pMGX127 IIa QKFSRLDPT 47 APS------SS (IIb) pMGX128 IIbKKFSRLDPT 48 APS------SS (IIb) pMGX129 IIa QKFSHLDPT 49 APS------SS(IIb) pMGX130 IIb KKFSHLDPT 50 APS------SS (IIb) pMGX131 IIa QKFSRLDPN51 VPSMGSSS(IIa) pMGX132 IIb KKFSRSDPN 52 VPSMGSSS(IIa) pMGX133RIIa-131R IIa QKFSRLDPT 53 VPSMGSSS(IIa) pMGX134 RIIa-131H IIa QKFSHLDPT54 VPSMGSSS(IIa) pMGX135 IIb KKFSRLDPT 55 VPSMGSSS(IIa) pMGX136 IIbKKFSHLDPT 56 VPSMGSSS(IIa)

The fusion proteins may be used in any biochemical assay fordetermination of binding to an anti-FcγRIIB antibody of the invention,e.g., an ELISA. In other embodiments, further confirmation of theepitope specificity may be done by using peptides with specific residuesreplaced with those from the FcγRIIA sequence.

The antibodies can be characterized using assays for identifying thefunction of the antibodies of the invention, particularly the activityto modulate FcγRIIB signaling. For example, characterization assays ofthe invention can measure phosphorylation of tyrosine residues in theITIM motif of FcγRIIB, or measure the inhibition of B cellreceptor-generated calcium mobilization. The characterization assays ofthe invention can be cell-based or cell-free assays.

It has been well established in the art that in mast cells coaggregationof FcγRIIB with the high affinity IgE receptor, FcεRI, leads toinhibition of antigen-induced degranulation, calcium mobilization, andcytokine production (Metcalfe D. D. et aL 1997, Physiol. Rev. 77:1033;Long E. O. 1999 Annu Rev. Immunol 17: 875). The molecular details ofthis signaling pathway have been recently elucidated (Ott V. L., 2002,J. Immunol. 162(9):4430-9). Once coaggregated with FcεRI, FcγRIIB israpidly phosphorylated on tyrosine in its ITIM motif, and then recruitsSrc Homology-2 containing inositol-5-phosphatase (SHIP), an SH2domain-containing inosital polyphosphate 5-phosphatase, which is in turnphosphorylated and associates with Shc and p62^(dok) (p62^(dok) is theprototype of a family of adaptor molecules, which includes signalingdomains such as an aminoterminal pleckstrin homology domain (PH domain),a PTB domain, and a carboxy terminal region containing PXXP motifs andnumerous phosphorylation sites (Carpino et al., 1997 Cell, 88: 197;Yamanshi et al., 1997, Cell, 88:205).

The anti-FcγRIIB antibodies for use in the invention may likewise becharacterized for ability to modulate one or more IgE mediatedresponses. Preferably, cells lines co-expressing the high affinityreceptor for IgE and the low affinity receptor for FcγRIIB will be usedin characterizing the anti-FcγRIIB antibodies in modulating IgE mediatedresponses. In a specific embodiment, cells from a rat basophilicleukemia cell line (RBL-H23; Barsumian E. L. et al. 1981 Eur. J.Immunol. 11:317, which is incorporated herein by reference in itsentirety) transfected with full length human FcγRIIB will be used.RBL-2H3 is a well characterized rat cell line that has been usedextensively to study the signaling mechanisms following IgE-mediatedcell activation. When expressed in RBL-2H3 cells and coaggregated withFcεRI, FcγRIIB inhibits FcεRI-induced calcium mobilization,degranulation, and cytokine production (Malbec et al., 1998, J. Immunol.160:1647; Daeron et al., 1995 J. Clin. Invest. 95:577; Ott et al., 2002J. of Immunol. 168:4430-4439).

Antibodies for use in the invention may also be characterized forinhibition of FcεRI induced mast cell activation. For example, cellsfrom a rat basophilic leukemia cell line (RBL-H23; Barsumian E. L. etal. 1981 Eur. J. Immunol. 11:317) that have been transfected withFcγRIIB are sensitized with IgE and stimulated either with F(ab′)₂fragments of rabbit anti-mouse IgG, to aggregate FcεRI alone, or withwhole rabbit anti-mouse IgG to coaggregate FcγRIIB and FcεRI. In thissystem, indirect modulation of down stream signaling molecules can beassayed upon addition of antibodies of the invention to the sensitizedand stimulated cells. For example, tyrosine phosphorylation of FcγRIIBand recruitment and phosphorylation of SHIP, activation of MAP kinasefamily members, including but not limited to Erk1, Erk2, JNK, or p38;and tyrosine phosphorylation of p62^(dok) and its association with SHIPand RasGAP can be assayed.

One exemplary assay for determining the inhibition of FcεRI induced mastcell activation by the antibodies of the invention can comprise of thefollowing: transfecting RBL-H23 cells with human FcγRIIB; sensitizingthe RBL-H23 cells with IgE; stimulating RBL-H23 cells with eitherF(ab′)₂ of rabbit anti-mouse IgG (to aggregate FcεRI alone and elicitFcεRI-mediated signaling, as a control), or stimulating RBL-H23 cellswith whole rabbit anti-mouse IgG to (to coaggregate FcγRIIB and FcεRI,resulting in inhibition of FcεRI-mediated signaling). Cells that havebeen stimulated with whole rabbit anti-mouse IgG antibodies can befurther pre-incubated with the antibodies of the invention. MeasuringFcεRI-dependent activity of cells that have been pre-incubated with theantibodies of the invention and cells that have not been pre-incubatedwith the antibodies of the invention, and comparing levels ofFcεRI-dependent activity in these cells, would indicate a modulation ofFcεRI-dependent activity by the antibodies of the invention.

The exemplary assay described above can be for example, used to identifyantibodies that block ligand (IgG) binding to FcγRIIB receptor andantagonize FcγRIIB-mediated inhibition of FcεRI signaling by preventingcoaggregating of FcγRIIB and FcεRI. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcεRI and agonizeFcγRIIB-mediated inhibition of FcεRI signaling by promotingcoaggregating of FcγRIIB and FcεRI.

In some embodiments, the anti-FcγRIIB diabodies, comprising the epitopebinding domains of anti-FcγRIIB antibodies identified described hereinor known in the art, of the invention are characterized for theirability to modulate an IgE mediated response by monitoring and/ormeasuring degranulation of mast cells or basophils, preferably in acell-based assay. Preferably, mast cells or basophils for use in suchassays have been engineered to contain human FcγRIIB using standardrecombinant methods known to one skilled in the art. In a specificembodiment the anti-FcγRIIB antibodies of the invention arecharacterized for their ability to modulate an IgE mediated response ina cell-based β-hexosaminidase (enzyme contained in the granules) releaseassay. β-hexosaminidase release from mast cells and basophils is aprimary event in acute allergic and inflammatory condition (Aketani etal., 2001 Immunol. Lett. 75: 185-9; Aketani et al., 2000 Anal. Chem. 72:2653-8). Release of other inflammatory mediators including but notlimited to serotonin and histamine may be assayed to measure an IgEmediated response in accordance with the methods of the invention.Although not intending to be bound by a particular mechanism of action,release of granules such as those containing β-hexosaminidase from mastcells and basophils is an intracellular calcium concentration dependentprocess that is initiated by the cross-linking of FcγRIs withmultivalent antigen.

The ability to study human mast cells has been limited by the absence ofsuitable long term human mast cell cultures. Recently two novel stemcell factor dependent human mast cell lines, designated LAD 1 and LAD2,were established from bone marrow aspirates from a patient with mastcell sarcoma/leukemia (Kirshenbaum et al., 2003, Leukemia research,27:677-82, which is incorporated herein by reference in its entirety.).Both cell lines have been described to express FcεRI and several humanmast cell markers. LAD 1 and 2 cells can be used for assessing theeffect of the antibodies of the invention on IgE mediated responses. Ina specific embodiment, cell-based β-hexosaminidase release assays suchas those described supra may be used in LAD cells to determine anymodulation of the IgE-mediated response by the anti-FcγRIIB antibodiesof the invention. In an exemplary assay, human mast cells, e.g., LAD 1,are primed with chimeric human IgE anti-nitrophenol (NP) and challengedwith BSA-NP, the polyvalent antigen, and cell degranulation is monitoredby measuring the β-hexosaminidase released in the supernatant(Kirshenbaum et al., 2003, Leukemia research, 27:677-682, which isincorporated herein by reference in its entirety).

In some embodiments, if human mast cells have a low expression ofendogenous FcγRIIB, as determined using standard methods known in theart, e.g., FACS staining, it may be difficult to monitor and/or detectdifferences in the activation of the inhibitory pathway mediated by theanti-FcγRIIB diabodies of the invention. The invention thus encompassesalternative methods, whereby the FcγRIIB expression may be upregulatedusing cytokines and particular growth conditions. FcγRIIB has beendescribed to be highly up-regulated in human monocyte cell lines, e.g.,THP1 and U937, (Tridandapani et al., 2002, J. Biol. Chem., 277(7):5082-5089) and in primary human monocytes (Pricop et al., 2001, J. ofImmunol., 166: 531-537) by IL4. Differentiation of U937 cells withdibutyryl cyclic AMP has been described to increase expression of FcγRII(Cameron et al., 2002 Immunology Letters 83, 171-179). Thus theendogenous FcγRIIB expression in human mast cells for use in the methodsof the invention may be up-regulated using cytokines, e.g., IL-4, IL-13,in order to enhance sensitivity of detection.

The anti-FcγRIIB diabodies can also be assayed for inhibition of B-cellreceptor (BCR)-mediated signaling. BCR-mediated signaling can include atleast one or more down stream biological responses, such as activationand proliferation of B cells, antibody production, etc. Coaggregation ofFcγRIIB and BCR leads to inhibition of cell cycle progression andcellular survival. Further, coaggregation of FcγRIIB and BCR leads toinhibition of BCR-mediated signaling.

Specifically, BCR-mediated signaling comprises at least one or more ofthe following: modulation of down stream signaling molecules (e.g.,phosphorylation state of FcγRIIB, SHIP recruitment, localization of Btkand/or PLCγ, MAP kinase activity, recruitment of Akt (anti-apoptoticsignal), calcium mobilization, cell cycle progression, and cellproliferation.

Although numerous effector functions of FcγRIIB-mediated inhibition ofBCR signaling are mediated through SHIP, recently it has beendemonstrated that lipopolysaccharide (LPS)-activated B cells from SHIPdeficient mice exhibit significant FcγRIIB-mediated inhibition ofcalcium mobilization, Ins(1,4,5)P₃ production, and Erk and Aktphosphorylation (Brauweiler A. et al., 2001, Journal of Immunology,167(1): 204-211). Accordingly, ex vivo B cells from SHIP deficient micecan be used to characterize the antibodies of the invention. Oneexemplary assay for determining FcγRIIB-mediated inhibition of BCRsignaling by the antibodies of the invention can comprise the following:isolating splenic B cells from SHIP deficient mice, activating saidcells with lipopolysachharide, and stimulating said cells with eitherF(ab′)₂ anti-IgM to aggregate BCR or with anti-IgM to coaagregate BCRwith FcγRIIB. Cells that have been stimulated with intact anti-IgM tocoaggregate BCR with FcγRIIB can be further pre-incubated with theantibodies of the invention. FcγRIIB-dependent activity of cells can bemeasured by standard techniques known in the art. Comparing the level ofFcγRIIB-dependent activity in cells that have been pre-incubated withthe antibodies and cells that have not been pre-incubated, and comparingthe levels would indicate a modulation of FcγRIIB-dependent activity bythe antibodies.

Measuring FcγRIIB-dependent activity can include, for example, measuringintracellular calcium mobilization by flow cytometry, measuringphosphorylation of Akt and/or Erk, measuring BCR-mediated accumulationof PI(3,4,5)P₃, or measuring FcγRIIB-mediated proliferation B cells.

The assays can be used, for example, to identify diabodies oranti-FcγRIIB antibodies for use in the invention that modulateFcγRIIB-mediated inhibition of BCR signaling by blocking the ligand(IgG) binding site to FcγRIIB receptor and antagonizing FcγRIIB-mediatedinhibition of BCR signaling by preventing coaggregation of FcγRIIB andBCR. The assays can also be used to identify antibodies that enhancecoaggregation of FcγRIIB and BCR and agonize FcγRIIB-mediated inhibitionof BCR signaling.

The anti-FcγRIIB antibodies can also be assayed for FcγRII-mediatedsignaling in human monocytes/macrophages. Coaggregation of FcγRIIB witha receptor bearing the immunoreceptor tyrosine-based activation motif(ITAM) acts to down-regulate FcγR-mediated phagocytosis using SHIP asits effector (Tridandapani et al. 2002, J. Biol. Chem. 277(7):5082-9).Coaggregation of FcγRIIA with FcγRIIB results in rapid phosphorylationof the tyrosine residue on FcγRIIB's ITIM motif, leading to anenhancement in phosphorylation of SHIP, association of SHIP with Shc,and phosphorylation of proteins having the molecular weight of 120 and60-65 kDa. In addition, coaggregation of FcγRIIA with FcγRIIB results indown-regulation of phosphorylation of Akt, which is a serine-threoninekinase that is involved in cellular regulation and serves to suppressapoptosis.

The anti-FcγRIIB diabodies can also be assayed for inhibition ofFcγR-mediated phagocytosis in human monocytes/macrophages. For example,cells from a human monocytic cell line, THP-1 can be stimulated eitherwith Fab fragments of mouse monoclonal antibody IV.3 against FcγRII andgoat anti-mouse antibody (to aggregate FcγRIIA alone), or with wholeIV.3 mouse monoclonal antibody and goat anti-mouse antibody (tocoaggregate FcγRIIA and FcγRIIB). In this system, modulation of downstream signaling molecules, such as tyrosine phosphorylation of FcγRIIB,phosphorylation of SHIP, association of SHIP with Shc, phosphorylationof Akt, and phosphorylation of proteins having the molecular weight of120 and 60-65 kDa can be assayed upon addition of molecules of theinvention to the stimulated cells. In addition, FcγRIIB-dependentphagocytic efficiency of the monocyte cell line can be directly measuredin the presence and absence of the antibodies of the invention.

Another exemplary assay for determining inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages by the antibodies of theinvention can comprise the following: stimulating THP-1 cells witheither Fab of IV.3 mouse anti-FcγRII antibody and goat anti-mouseantibody (to aggregate FcγRIIA alone and elicit FcγRIIA-mediatedsignaling); or with mouse anti-FcγRII antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB and inhibitingFcγRIIA-mediated signaling. Cells that have been stimulated with mouseanti-FcγRII antibody and goat anti-mouse antibody can be furtherpre-incubated with the molecules of the invention. MeasuringFcγRIIA-dependent activity of stimulated cells that have beenpre-incubated with molecules of the invention and cells that have notbeen pre-incubated with the antibodies of the invention and comparinglevels of FcγRIIA-dependent activity in these cells would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention.

The exemplary assay described can be used for example, to identifybinding domains that block ligand binding of FcγRIIB receptor andantagonize FcγRIIB-mediated inhibition of FcγRIIA signaling bypreventing coaggregation of FcγRIIB and FcγRIIA. This assay likewiseidentifies binding domains that enhance coaggregation of FcγRIIB andFcγRIIA and agonize FcγRIIB-mediated inhibition of FcγRIIA signaling.

The FcγRIIB binding domains of interest can be assayed while comprised Iantibodies by measuring the ability of THP-1 cells to phagocytosefluoresceinated IgG-opsonized sheep red blood cells (SRBC) by methodspreviously described (Tridandapani et al., 2000, J. Biol. Chem. 275:20480-7). For example, an exemplary assay for measuring phagocytosiscomprises of: treating THP-1 cells with the antibodies of the inventionor with a control antibody that does not bind to FcγRII, comparing theactivity levels of said cells, wherein a difference in the activities ofthe cells (e.g., rosetting activity (the number of THP-1 cells bindingIgG-coated SRBC), adherence activity (the total number of SRBC bound toTHP-1 cells), and phagocytic rate) would indicate a modulation ofFcγRIIA-dependent activity by the antibodies of the invention. Thisassay can be used to identify, for example, antibodies that block ligandbinding of FcγRIIB receptor and antagonize FcγRIIB-mediated inhibitionof phagocytosis. This assay can also identify antibodies that enhanceFcγRIIB-mediated inhibition of FcγRIIA signaling.

In a preferred embodiment, the binding domains modulateFcγRIIB-dependent activity in human monocytes/macrophages in at leastone or more of the following ways: modulation of downstream signalingmolecules (e.g., modulation of phosphorylation state of FcγRIIB,modulation of SHIP phosphorylation, modulation of SHIP and Shcassociation, modulation of phosphorylation of Akt, modulation ofphosphorylation of additional proteins around 120 and 60-65 kDa) andmodulation of phagocytosis.

5.1.2 CD16A Binding Domains

The following section discusses CD16A binding proteins which can be usedas sources for light and heavy chain variable regions for covalentdiabody production. In the present invention CD16A binding proteinsincludes molecules comprising VL and VH domains of anti-CD16Aantibodies, which VH and VL domains are used in the production of thediabodies of the present invention.

A variety of CD16A binding proteins may be used in connection with thepresent invention. Suitable CD16A binding proteins include human orhumanized monoclonal antibodies as well as CD16A binding antibodyfragments (e.g., scFv or single chain antibodies, Fab fragments,minibodies) and another antibody-like proteins that bind to CD16A via aninteraction with a light chain variable region domain, a heavy chainvariable region domain, or both.

In some embodiments, the CD16A binding protein for use according to theinvention comprises a VL and/or VH domain that has one or more CDRs withsequences derived from a non-human anti-CD16A antibody, such as a mouseanti-CD16A antibody, and one or more framework regions with derived fromframework sequences of one or more human immunoglobulins. A number ofnon-human anti-CD16A monoclonal antibodies, from which CDR and othersequences may be obtained, are known (see, e.g., Tamm and Schmidt, 1996,J. Imm. 157:1576-81; Fleit et al., 1989, p. 159; LEUKOCYTE TYPING II:HUMAN MYELOID AND HEMATOPOIETIC CELLS, Reinherz et al., eds. New York:Springer-Verlag; 1986; LEUCOCYTE TYPING III: WHITE CELL DIFFERENTIATIONANTIGENS McMichael A J, ed., Oxford: Oxford University Press, 1986);LEUKOCYTE TYPING IV: WHITE CELL DIFFERENTIATION ANTIGENS, Kapp et al.,eds. Oxford Univ. Press, Oxford; LEUKOCYTE TYPING V: WHITE CELLDIFFERENTIATION ANTIGENS, Schlossman et al., eds. Oxford Univ. Press,Oxford; LEUKOCYTE TYPING VI: WHITE CELL DIFFERENTIATION ANTIGENS,Kishimoto, ed. Taylor & Francis. In addition, as shown in the Examples,new CD16A binding proteins that recognize human CD16A expressed on cellscan be obtained using well known methods for production and selection ofmonoclonal antibodies or related binding proteins (e.g., hybridomatechnology, phage display, and the like). See, for example, O'Connel etal., 2002, J. Mol. Biol. 321:49-56; Hoogenboom and Chames, 2000, Imm.Today 21:371078; Krebs et al., 2001, J. Imm. Methods 254:67-84; andother references cited herein. Monoclonal antibodies from a non-humanspecies can be chimerized or humanized using techniques using techniquesof antibody humanization known in the art.

Alternatively, fully human antibodies against CD16A can be producedusing transgenic animals having elements of a human immune system (see,e.g., U.S. Pat. Nos. 5,569,825 and 5,545,806), using human peripheralblood cells (Casali et al., 1986, Science 234:476), by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse et al., 1989, Science 246:1275, and by other methods.

In a preferred embodiment, the binding donor is from the 3G8 antibody ora humanized version thereof, e.g., such as those disclosed in U.S.patent application publication 2004/0010124, which is incorporated byreference herein in its entirety. It is contemplated that, for somepurposes, it may be advantageous to use CD16A binding proteins that bindthe CD16A receptor at the same epitope bound by 3G8, or at leastsufficiently close to this epitope to block binding by 3G8. Methods forepitope mapping and competitive binding experiments to identify bindingproteins with the desired binding properties are well known to thoseskilled in the art of experimental immunology. See, for example, Harlowand Lane, cited supra; Stahl et al., 1983, Methods in Enzymology9:242-53; Kirkland et al., 1986, J. Immunol. 137:3614-19; Morel et al.,1988, Molec. Immunol. 25:7-15; Cheung et al., 1990, Virology 176:546-52;and Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82. For instance,it is possible to determine if two antibodies bind to the same site byusing one of the antibodies to capture the antigen on an ELISA plate andthen measuring the ability of the second antibody to bind to thecaptured antigen. Epitope comparison can also be achieved by labeling afirst antibody, directly or indirectly, with an enzyme, radionuclide orfluorophore, and measuring the ability of an unlabeled second antibodyto inhibit the binding of the first antibody to the antigen on cells, insolution, or on a solid phase.

It is also possible to measure the ability of antibodies to block thebinding of the CD16A receptor to immune complexes formed on ELISAplates. Such immune complexes are formed by first coating the plate withan antigen such as fluorescein, then applying a specificanti-fluorescein antibody to the plate. This immune complex then servesas the ligand for soluble Fc receptors such as sFcRIIIa. Alternatively asoluble immune complex may be formed and labeled, directly orindirectly, with an enzyme radionuclide or fluorophore. The ability ofantibodies to inhibit the binding of these labeled immune complexes toFc receptors on cells, in solution or on a solid phase can then bemeasured.

CD16A binding proteins of the invention may or may not comprise a humanimmunoglobulin Fc region. Fc regions are not present, for example, inscFv binding proteins. Fc regions are present, for example, in human orhumanized tetrameric monoclonal IgG antibodies. As described supra, insome embodiments of the present invention, the CD 16A binding proteinincludes an Fc region that has an altered effector function, e.g.,reduced affinity for an effector ligand such as an Fc receptor or C1component of complement compared to the unaltered Fc region (e.g., Fc ofnaturally occurring IgG1, proteins). In one embodiment the Fc region isnot glycosylated at the Fc region amino acid corresponding to position297. Such antibodies lack Fc effector function.

Thus, the CD16A binding protein may not exhibit Fc-mediated binding toan effector ligand such as an Fc receptor or the C1 component ofcomplement due to the absence of the Fc domain in the binding proteinwhile, in other cases, the lack of binding or effector function is dueto an alteration in the constant region of the antibody.

5.1.2.1 CD16A Binding Proteins Comprising CDR Sequences Similar to a mAb3G8 CDR Sequences.

CD16A binding proteins that can be used in the practice of the inventioninclude proteins comprising a CDR sequence derived from (i.e., having asequence the same as or similar to) the CDRs of the mouse monoclonalantibody 3G8. Complementary cDNAs encoding the heavy chain and lightchain variable regions of the mouse 3G8 monoclonal antibody, includingthe CDR encoding sequences, were cloned and sequenced as described. Thenucleic acid and protein sequences of 3G8 are provided below. Using themouse variable region and CDR sequences, a large number of chimeric andhumanized monoclonal antibodies, comprising complementary determiningregions derived from 3G8 CDRs were produced and their propertiesanalyzed. To identify humanized antibodies that bind CD16A with affinityand have other desirable properties, antibody heavy chains comprising aVH region with CDRs derived from 3G8 were produced and combined (bycoexpression) with antibody light chains comprising a VL region withCDRs derived from 3G8 to produce a tetrameric antibody for analysis.Properties of the resulting tetrameric antibodies were determined asdescribed below. As described below, CD16A binding proteins comprising3G8 CDRs, such as the humanized antibody proteins described herein, maybe used according to the invention.

5.1.2.1.1 VH Region

In one aspect, the CD16A binding protein of the invention may comprise aheavy chain variable domain in which at least one CDR (and usually threeCDRS) have the sequence of a CDR (and more typically all three CDRS) ofthe mouse monoclonal antibody 3G8 heavy chain for which the remainingportions of the binding protein are substantially human (derived fromand substantially similar to, the heavy chain variable region of a humanantibody or antibodies).

In an aspect, the invention provides a humanized 3G8 antibody orantibody fragment containing CDRs derived from the 3G8 antibody in asubstantially human framework, but in which at least one of the CDRs ofthe heavy chain variable domain differs in sequence from thecorresponding mouse antibody 3G8 heavy chain CDR. For example, in oneembodiment, the CDR(S) differs from the 3G8 CDR sequence at least byhaving one or more CDR substitutions shown known in the art to affectbinding of 3G8 to CD16A, as known in the art or as disclosed in Tables 3and 4A-H. Suitable CD16 binding proteins may comprise 0, 1, 2, 3, or 4,or more of these substitutions (and often have from 1 to 4 of thesesubstitutions) and optionally can have additional substitutions as well.TABLE 3 V_(H) Domain Substitutions Kabat No. Position RegionSubstitutions 1 2 FR1 Ile 2 5 FR1 Lys 3 10 FR1 Thr 4 30 FR1 Arg 5 34CDR1 Val 6 50 CDR2 Leu 7 52 CDR2 Phe or Tyr or Asp 8 54 CDR2 Asn 9 60CDR2 Ser 10 62 CDR2 Ser 11 70 FR3 Thr 12 94 FR3 Gln or Lys or Ala or His13 99 CDR3 Tyr 14 101 CDR3 Asp

TABLE 4A V_(H) Sequences Derived from 3G8 V_(H)* FR1 CDR1 FR2 CDR2 FR3CDR3 FR4 3G8VH A A A A A A A Ch3G8VH A A A A A A B HxC B A B A A A B CxHA A A A B A B Hu3G8VH-1 B A B A B A B Hu3G8VH-2 C A B A B A B Hu3G8VH-3D A B A B A B Hu3G8VH-4 B A B A C B B Hu3G8VH-5 B A B A C A B Hu3G8VH-6B B B A B B B Hu3G8VH-7 B B B A B A B Hu3G8VH-8 B A B A B C B Hu3G8VH-9B A B B B B B Hu3G8VH-10 B A B A B B B Hu3G8VH-11 B A B B B A BHu3G8VH-12 B A B C B A B Hu3G8VH-13 B A B D B A B Hu3G8VH-14 B A B E B AB Hu3G8VH-15 B A B A D A B Hu3G8VH-16 B A B A E A B Hu3G8VH-17 B A B A FA B Hu3G8VH-18 B A B A G A B Hu3G8VH-19 B A B A C C B Hu3G8VH-20 B B B CB A B Hu3G8VH-21 B A B A D B B Hu3G8VH-22 B B B C B C B Hu3G8VH-23 B B BC E C B Hu3G8VH-24 B B B C F C B Hu3G8VH-25 B B B C G C B Hu3G8VH-26 B BB C C C B Hu3G8VH-27 B B B C E D B Hu3G8VH-28 B B B C F D B Hu3G8VH-29 BB B C G D B Hu3G8VH-30 B B B C C D B Hu3G8VH-31 E B B C B A B Hu3G8VH-32E B B H B A B Hu3G8VH-33 E B B H B A B Hu3G8VH-34 E B B C B C BHu3G8VH-35 E B B C C C B Hu3G8VH-36 E B B H C D B Hu3G8VH-37 E B B H E CB Hu3G8VH-38 E B B F B A B Hu3G8VH-39 E B B I B A B Hu3G8VH-40 E B B G BA B Hu3G8VH-41 E B B J B A B Hu3G8VH-42 E B B C H A B Hu3G8VH-43 E B B CH C B Hu3G8VH-44 E B B C I D B Hu3G8VH-45 E B B C J D B*Letters in Table 4A refer to sequences in Tables 4 B-H.

TABLE 4B FR1 A B C D E RESIDUE Q Q Q Q Q 1 V V V V I 2 T T T T T 3 L L LL L 4 K R K R K 5 E E E E E 6 S S S S S 7 G G G G G 8 P P P P P 9 G A AA T 10 I L L L L 11 L V V V V 12 Q K K K K 13 P P P P P 14 S T T T T 15Q Q Q Q Q 16 T T T T T 17 L L L L L 18 S T T T T 19 L L L L L 20 T T T TT 21 C C C C C 22 S T T T T 23 F F F F F 24 S S S S S 25 G G G G G 26 FF F F F 27 S S S S S 28 L L L L L 29 R S S R S 30 30 31 32 33 34 Se IDNo

TABLE 4C CDR1 A B RESIDUE T T 31 S S 32 G G 33 M V 34 G G 35 V V 35A G G35B 35 36 Seq ID No

TABLE 4D FR2 A B RESIDUE W W 36 I I 37 R R 38 Q Q 39 P P 40 S P 41 G G42 K K 43 G A 44 L L 45 E E 46 W W 47 L L 48 A A 49 37 38 Seq ID No.

TABLE 4E CDR2 A B C D E F G H I J RESIDUE H H H H H L H L H L 50 I I I 1I I I I I I 51 W Y W Y W D F W D W 52 W W W W W W W W W W 53 D N D D N DD D D N 54 D D D D D D D D D D 55 D D D D D D D D D D 56 K K K K K K K KK K 57 R R R R R R R R R R 58 Y Y Y Y Y Y Y Y Y Y 59 N N S N N S S S S S60 P P P P P P P P P P 61 A A S A A S S S S S 62 L L L L L L L L L L 63K K K K K K K K K K 64 S S S S S S S S S S 65 39 40 41 42 43 44 45 46 4748 Seq ID No

TABLE 4F FR3 A B C D E F G H I J RESIDUE R R R R R R R R R R 66 L L L LL L L L L L 67 I T T T T T T T T T 68 I I I I I I I I I I 69 S S S S S SS T T T 70 K K K K K K K K K K 71 D D D D D D D D D D 72 T T T T T T T IT T 73 S S S S S S S S S S 74 S K K K K K K K K K 75 N N N N N N N N N N76 Q Q Q Q Q Q Q Q Q Q 77 V V V V V V V V V V 78 F V V V V V V V V V 79L L L L L L L L L L 80 K T T T T T T T T T 81 I M M M M M M M M M 82 A TT T T T T T T T 82A S N N N N N N N N N 82B V M M M M M M M M M 82C D DD D D D D D D D 83 T P P P P P P P P P 84 A V V V V V V V V V 85 D D D DD D D D D D 86 T T T T T T T T T T 87 A A A A A A A A A A 88 T T T T T TT T T T 89 Y Y Y Y Y Y Y Y Y Y 90 Y Y Y Y Y Y Y Y Y Y 91 C C C C C C C CC C 92 A A A A A A A A A A 93 Q R Q T K A H R H Q 94 49 50 51 52 53 5455 56 57 58 Seq ID No

TABLE 4G CDR3 A B C D RESIDUE I I I I 95 N N N N 96 P P P P 97 A A A A98 W W Y Y 99 F F F F 100 A D A D 101 Y Y Y Y 102 59 60 61 62 Seq ID No

TABLE 4H FR4 A B RESIDUE W W 103 G G 104 Q Q 105 G G 106 T T 107 L L 108V V 109 T T 110 V V 111 S S 112 A S 113 63 64 Seq ID No

In one embodiment, a CD16A binding protein may comprise a heavy chainvariable domain sequence that is the same as, or similar to, the VHdomain of the Hu3G8VH-1 construct, the sequence of which is provided inSEQ ID NO:70. For example, the invention provides a CD16A bindingprotein comprising a VH domain with a sequence that (1) differs from theVH domain of Hu3G8VH-1 (SEQ ID NO:70) by zero, one, or more than one ofthe CDR substitutions set forth in Table 1; (2) differs from the VHdomain of Hu3G8VH-1 by zero, one or more than one of the frameworksubstitutions set forth in Table 1; and (3) is at least about 80%identical, often at least about 90%, and sometimes at least about 95%identical, or even at least about 98% identical to the Hu3G8VH-1 VHsequence at the remaining positions.

Exemplary VH domains of CD16 binding proteins of the invention have thesequence of 3G8VH, Hu3G8VH-5 and Hu3G8VH-22 (SEQ ID NO:81, SEQ ID NO:71and SEQ ID NO:72, respectively)e. Examplary nucleotide sequencesencoding the sequences of 3G8VH and Hu3G8VH-5 (SEQ ID NO:81 and SEQ IDNO:71, respectively) are provided by SEQ ID NO:82 and SEQ ID NO:83,respectively.

The VH domain may have a sequence that differs from that of Hu3G8VH-1(SEQ ID NO:70) by at least one, at least two, at least three, at leastfour 4, at least five, or at least six of the substitutions shown inTable 3. These substitutions are believed to result in increasedaffinity for CD16A and/or reduce the immunogenicity of a CD16A bindingprotein when administered to humans. In certain embodiments, the degreeof sequence identity with the Hu3G8VH-1 VH domain at the remainingpositions is at least about 80%, at least about 90%, at least about 95%or at least about 98%.

For illustration and not limitation, the sequences of a number of CD16Abuilding protein VH domains is shown in Table 4. Heavy chains comprisingthese sequences fused to a human Cγ1 constant region were coexpressedwith the hu3G8VL-1 light chain (described below) to form tetramericantibodies, and binding of the antibodies to CD16A was measured toassess the effect of amino acid substitutions compared to the hu3G8VH-1VH domain. Constructs in which the VH domain has a sequence ofhu3G8VH-1, 2, 3, 4, 5, 8, 12, 14, 16, 17, 18, 19, 20, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 42, 43, 44 and 45 showedhigh affinity binding, with hu3G8VH-6 and −40 VH domains showingintermediate binding. CD16A binding proteins comprising the VH domainsof hu3G8VH-5 and hu3G8VH-22 (SEQ ID NO:71 and SEQ ID NO:72,respectively) are considered to have particularly favorable bindingproperties.

5.1.2.2 VL Region

Similar studies were conducted to identify light chain variable domainsequences with favorable binding properties. In one aspect, theinvention provides a CD16A binding protein containing a light chainvariable domain in which at least one CDR (and usually three CDRs) hasthe sequence of a CDR (and more typically all three CDRs) of the mousemonoclonal antibody 3G8 light chain and for which the remaining portionsof the binding protein are substantially human (derived from andsubstantially similar to, the heavy chain variable region of a humanantibody or antibodies).

In one aspect, the invention provides a fragment of a humanized 3G8antibody containing CDRs derived from the 3G8 antibody in asubstantially human framework, but in which at least one of the CDRs ofthe light chain variable domain differs in sequence from the mousemonoclonal antibody 3G8 light chain CDR. In one embodiment, the CDR(s)differs from the 3G8 sequence at least by having one or more amino acidsubstitutions in a CDR, such as, one or more substitutions shown inTable 2 (e.g., arginine at position 24 in CDR1; serine at position 25 inCDR1; tyrosine at position 32 in CDR1; leucine at position 33 in CDR1;aspartic acid, tryptophan or serine at position 50 in CDR2; serine atposition 53 in CDR2; alanine or glutamine at position 55 in CDR2;threonine at position 56 in CDR2; serine at position 93 in CDR3; and/orthreonine at position 94 in CDR3). In various embodiments, the variabledomain can have 0, 1, 2, 3, 4, 5, or more of these substitutions (andoften have from 1 to 4 of these substitutions) and optionally, can haveadditional substitutions as well.

In one embodiment, a suitable CD16A binding protein may comprise a lightchain variable domain sequence that is the same as, or similar to, theVL domain of the Hu3G8VL-1 (SEQ ID NO:73) construct, the sequence ofwhich is provided in Table 6. For example, the invention provides aCD16A binding protein comprising a VL domain with a sequence that (1)differs from the VL domain of Hu3G8VL-1 (SEQ ID NO:73) by zero, one, ormore of the CDR substitutions set forth in Table 5; (2) differs from theVL domain of Hu3G8VL-1 by zero, one or more of the frameworksubstitutions set forth in Table 5; and (3) is at least 80% identical,often at least about 90%, and sometimes at least about 95% identical, oreven at least about 98% identical to the Hu3G8VL-1 VL sequence (SEQ IDNO:73) at the remaining positions. TABLE 5 3G8 V_(L) DomainSubstitutions Kabat No. Position Region Substitutions 1 24 CDR1 Arg 2 25CDR1 Ser 3 32 CDR1 Tyr 4 33 CDR1 Leu 5 50 CDR2 Asp or Trp or Ser 6 51CDR2 Ala 7 53 CDR2 Ser 8 55 CDR2 Ala or Gln 9 56 CDR2 Thr 10 93 CDR3 Ser11 94 CDR3 Thr

TABLE 6 V_(L) Sequences Derived from 3G8 V_(L)* FR1 CDR1 FR2 CDR2 FR3CDR3 FR4 3G8VL A A A A A A A Ch3G8VL A A A A A A A Hu3G8VL-1 B A A A B AB Hu3G8VL-2 B B A A B A B Hu3G8VL-3 B C A A B A B Hu3G8VL-4 B D A A B AB Hu3G8VL-5 B E A A B A B Hu3G8VL-6 B F A A B A B Hu3G8VL-7 B G A A B AB Hu3G8VL-8 B A A B B A B Hu3G8VL-9 B A A C B A B Hu3G8VL- B A A D B A B10 Hu3G8VL- B A A E B A B 11 Hu3G8VL- B A A F B A B 12 Hu3G8VL- B A A GB A B 13 Hu3G8VL- B A A A B B B 14 Hu3G8VL- B A A A B C B 15 Hu3G8VL- BA A A B D B 16 Hu3G8VL- B A A A B E B 17 Hu3G8VL- B B A D B A B 18Hu3G8VL- B B A D B D B 19 Hu3G8VL- B B A D B E B 20 Hu3G8VL- B C A D B AB 21 Hu3G8VL- B C A D B D B 22 Hu3G8VL- B C A D B E B 23 Hu3G8VL- B D AD B A B 24 Hu3G8VL- B D A D B D B 25 Hu3G8VL- B D A D B E B 26 Hu3G8VL-B E A D B A B 27 Hu3G8VL- B E A D B D B 28 Hu3G8VL- B E A D B E B 29Hu3G8VL- B A A D B D B 30 Hu3G8VL- B A A D B E B 31 Hu3G8VL- B A A H B AB 32 Hu3G8VL- B A A I B A B 33 Hu3G8VL- B A A J B A B 34 Hu3G8VL- B B AH B D B 35 Hu3G8VL- B C A H B D B 36 Hu3G8VL- B E A H B D B 37 Hu3G8VL-B B A I B D B 38 Hu3G8VL- B C A I B D B 39 Hu3G8VL- B E A I B D B 40Hu3G8VL- B B A J B D B 41 Hu3G8VL- B C A J B D B 42 Hu3G8VL- B E A J B DB 43 Hu3G8VL- B A A K B A B 44*Letters in Table 6A refer to sequences in Tables 6B-H.

TABLE 6B FR1 A B RESIDUE D D 1 T I 2 V V 3 L M 4 T T 5 Q Q 6 S S 7 P P 8A D 9 S S 10 L L 11 A A 12 V V 13 S S 14 L L 15 G G 16 Q E 17 R R 18 A A19 T T 20 I I 21 S N 22 C C 23 65 66 Seq ID No

TABLE 6C CDR1 A B C D E F G RESIDUE K R K K K K K 24 A A S A A A A 25 SS S S S S S 26 Q Q Q Q Q Q Q 27 S S S S S S S 27A V V V V V V V 27B D DD D D D D 27C F F F F F F F 27D D D D D D D D 28 G G G G G G G 29 D D DD D D D 30 S S S S S S S 31 F F F Y F F Y 32 M M M M L M L 33 N N N N NA A 34 67 68 69 70 71 72 73 Seq ID No

TABLE 6D FR2 A RESIDUE W 35 Y 36 Q 37 Q 38 K 39 P 40 G 41 Q 42 P 43 P 44K 45 L 46 L 47 I 48 Y 49 74 Seq ID No

TABLE 6E CDR2 A B C D E F G H I J K RESIDUE T D W T D D S S S T T 50 T AA T A A A T T T T 51 S S S S S S S S S S S 52 N N N N N N N N N N S 53 LL L L L L L L L L L 54 E E E E E A Q E Q Q Q 55 S S S T T T S S S S S 5675 76 77 78 79 80 81 82 83 84 85 Seq ID No

TABLE 6F FR3 A B RESIDUE G G 57 I V 58 P P 59 A D 60 R R 61 F F 62 S S63 A G 64 S S 65 G G 66 S S 67 G G 68 T T 69 D D 70 F F 71 T T 72 L L 73N T 74 I I 75 H S 76 P S 77 V L 78 E Q 79 E A 80 E E 81 D D 82 T V 83 AA 84 T V 85 Y Y 86 Y Y 87 C C 88 86 87 Seq ID No

TABLE 6G CDR3 A B C D E RESIDUE Q Q Q Q Q 89 Q Q Q Q Q 90 S S S S S 91 NY Y N N 92 E S E S E 93 D T D D T 94 P P P P P 95 Y Y Y Y Y 96 T T T T T97 88 89 90 91 92 Seq ID No

TABLE 6H FR4 A B RESIDUE F F 98 G G 99 G Q 100 G G 101 T T 102 K K 103 LL 104 E E 105 I I 106 K K 107 93 94 Seq D No

Exemplary VL domains of CD16 binding proteins of the invention have thesequence of 3G8VL, Hu3G8VL-1 or Hu3G8VL-43, (SEQ ID NO:84, SEQ ID NO:73and SEQ ID NO:74, respectively) as shown in Tables 5 and 6. Exemplarynucleotide sequences encoding 3G8VL (SEQ ID NO:84) and Hu3G8VL-1 (SEQ IDNO:73) are provided in SEQ ID NO:85 and SEQ ID NO:86, respectively.

The VL domain may have a sequence that differs from that of Hu3G8VL-1(SEQ ID NO:73) by zero, one, at least two, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, or at least 9 of thesubstitutions shown in Table 2. These substitutions are believed toresult in increased affinity for CD16A and/or reduce the immunogenicityof a CD16A binding protein when administered to humans. In certainembodiments, the degree of sequence identity at the remaining positionsis at least about 80%, at least about 90% at least about 95% or at leastabout 98%.

For illustration and not limitation, the sequences of a number of CD16Abinding proteins VL domains is shown in Table 6. Light chains comprisingthese sequences fused to a human Cκ. constant domain were coexpressedwith a Hu3G8VH heavy chain (described above) to form tetramericantibodies, and the binding of the antibodies to CD16A was measured toassess the effect of amino acid substitutions compared to the Hu3G8VL-1VL domain (SEQ ID NO:73). Constructs in which the VL domain has asequence of hu3G8VL-1, 2, 3, 4, 5, 10, 16, 18, 19, 21, 22, 24, 27, 28,32, 33, 34, 35, 36, 37, and 42 showed high affinity binding andhu3G8VL-15, 17, 20, 23, 25, 26, 29, 30, 31, 38, 39, 40 and 41 showedintermediate binding. CD16A binding proteins comprising the VL domainsof hu3G8VL-1, hu3G8VL-22, and hu3G8VL-43 are considered to haveparticularly favorable binding properties (SEQ ID NO:73, SEQ ID NO:75and SEQ ID NO:74, respectively).

5.1.2.2.1 Combinations of VL and/or VH Domains

As is known in the art and described elsewhere herein, immunoglobulinlight and heavy chains can be recombinantly expressed under conditionsin which they associate to produce a diabody, or can be so combined invitro. It will thus be appreciated that a 3G8-derived VL-domaindescribed herein can be combined a 3G8-derived VH-domain describedherein to produce a CD16A binding diabody, and all such combinations arecontemplated.

For illustration and not for limitation, examples of useful CD16Adiabodies are those comprising at least one VH domain and at least oneVL domain, where the VH domain is from hu3G8VH-1, hu3G8VH-22 orhu3G8VH-5 (SEQ ID NO:70, SEQ ID NO:72 and SEQ ID NO:71, respectively)and the VL domain is from hu3G8VL-1, hu3G8VL-22 or hu3G8VL-43 (SEQ IDNO:73, SEQ ID NO:75 and SEQ ID NO:43, respectively). In particular,humanized antibodies that comprise hu3G8VH-22 (SEQ ID NO:22) and either,hu3G8VL-1, hu3G8VL-22 or hu3G8VL-43 (SEQ ID NO:73, SEQ ID NO:72 and SEQID NO:74, respectively), or hu3G8VH-5 (SEQ ID NO:71) and hu3G8VL-1 (SEQID NO:73) have favorable properties.

It will be appreciated by those of skill that the sequences of VL and VHdomains described here can be further modified by art-known methods suchas affinity maturation (see Schier et al., 1996, J. Mol. Biol.263:551-67; Daugherty et al., 1998, Protein Eng. 11:825-32; Boder etal., 1997, Nat. Biotechnol. 15:553-57; Boder et al., 2000, Proc. Natl.Acad. Sci. U.S.A 97:10701-705; Hudson and Souriau, 2003, Nature Medicine9:129-39). For example, the CD16A binding proteins can be modified usingaffinity maturation techniques to identify proteins with increasedaffinity for CD16A and/or decreased affinity for CD16B.

One exemplary CD16 binding protein is the mouse 3G8 antibody. Amino acidsequence comprising the VH and VL domains of humanized 3G8 are describedin FIGS. 2, 9, 14 and set forth in SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74.

5.2 Diabodies Comprising Fc Regions or Portions thereof

The invention encompasses diabody molecules comprising Fc domains orportions thereof (e.g., a CH2 or CH3 domain). In certain embodiments theFc domain, or portion(s) thereof, comprises one or more constantdomain(s) of the Fc region of IgG2, IgG3 or IgG4 (e.g., CH2 or CH3). Inother embodiments, the invention encompasses molecules comprising and Fcdomain or portion therof, wherein said Fc domain or portion thereofcomprises at least one amino acid modification (e.g. substitution)relative to a comparable wild-type Fc domain or portion thereof. VariantFc domains are well known in the art, and are primarily used to alterthe phenotype of the antibody comprising said variant Fc domain asassayed in any of the binding activity or effector function assays wellknown in the art, e.g. ELISA, SPR analysis, or ADCC. Such variant Fcdomains, or portions thereof, have use in the present invention byconferring or modifying the effector function exhibited by a diabodymolecule of the invention comprising an Fc domain (or portion thereof)as functionally assayed, e.g., in an NK dependent or macrophagedependent assay. Fc domain variants identified as altering effectorfunction are disclosed in International Application WO04/063351, U.S.Patent Application Publications 2005/0037000 and 2005/0064514, U.S.Provisional Applications 60/626,510, filed Nov. 10, 2004, 60/636,663,filed Dec. 15, 2004, and 60/781,564, filed Mar. 10, 2006, and U.S.patent applications Ser. No. 11/271,140, filed Nov. 10, 2005, and Ser.No. 11/305,787, filed Dec. 15, 2005, concurrent applications of theInventors, each of which is incorporated by reference in its entirety.

In other embodiments, the invention encompasses the use of any Fcvariant known in the art, such as those disclosed in Duncan et al, 1988,Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund etal, 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund etal., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett54:101-104; Lund et al, 1996, J Immunol 157:49634969; Armour et al,1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol164:41784184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu etal.,2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); US 5,624,821; US 5,885,573; US 6,194,551; PCT WO00/42072; PCT WO 99/58572; each of which is incorporated herein byreference in its entirety.

In certain embodiments, said one or more modifications to the aminoacids of the Fc region reduce the affinity and avidity of the Fc regionand, thus, the diabody molecule of the invention, for one or more FcγRreceptors. In a specific embodiment, the invention encompasses diabodiescomprising a variant Fc region, or portion thereof, wherein said variantFc region comprises at least one amino acid modification relative to awild type Fc region, which variant Fc region only binds one FcγR,wherein said FcγR is FcγRIIIA. In another specific embodiment, theinvention encompasses diabodies comprising a variant Fc region, orportion thereof, wherein said variant Fc region comprises at least oneamino acid modification relative to a wild type Fc region, which variantFc region only binds one FcγR, wherein said FcγR is FcγRIIA. In anotherspecific embodiment, the invention encompasses diabodies comprising avariant Fc region, or portion thereof, wherein said variant Fc regioncomprises at least one amino acid modification relative to a wild typeFc region, which variant Fc region only binds one FcγR, wherein saidFcγR is FcγRIIB. In certain embodiments, the invention encompassesmolecules comprising a variant Fc domain wherein said variant confers ormediates increased ADCC activity and/or an increased binding to FcγRIIA(CD32A), relative to a molecule comprising no Fc domain or comprising awild-type Fc domain, as measured using methods known to one skilled inthe art and described herein. In alternate embodiments, the inventionencompasses molecules comprising a variant Fc domain wherein saidvariant confers or mediates decreased ADCC activity (or other effectorfunction) and/or an increased binding to FcγRIIB (CD32B), relative to amolecule comprising no Fc domain or comprising a wild-type Fc domain, asmeasured using methods known to one skilled in the art and describedherein.

The invention also encompasses the use of an Fc domain comprisingdomains or regions from two or more IgG isotypes. As known in the art,amino acid modification of the Fc region can profoundly affectFc-mediated effector function and/or binding activity. However, thesealterations in functional characteristics can be further refined and/ormanipulated when implemented in the context of selected IgG isotypes.Similarly, the native characteristics of the isotype Fc may bemanipulated by the one or more amino acid modifications. The multipleIgG isotypes (i.e., IgG1, IgG2, IgG3 and IgG4) exhibit differingphysical and functional properties including serum half-life, complementfixation, FcγR binding affinities and effector function activities (e.g.ADCC, CDC) due to differences in the amino acid sequences of their hingeand/or Fc domains. In certain embodiments, the amino acid modificationand IgG Fc region are independently selected based on their respective,separate binding and/or effector function activities in order toengineer a diabody with desired characteristics. In most embodiments,said amino acid modifications and IgG hinge/Fc regions have beenseparately assayed for binding and/or effector function activity asdescribed herein or known in the art in an the context of an IgG1. Incertain embodiments, said amino acid modification and IgG hinge/Fcregion display similar functionality, e.g., increased affinity forFcγRIIA, when separately assayed for FcγR binding or effector functionin the context of the diabody molecule or other Fc-containing molecule(e.g. and immunoglobulin). The combination of said amino acidmodification and selected IgG Fc region then act additively or, morepreferably, synergistically to modify said functionality in the diabodymolecule of the invention, relative to a diabody molecule of theinvention comprising a wild-type Fc region. In other embodiments, saidamino acid modification and IgG Fc region display oppositefunctionalities, e.g., increased and decreased, respectively, affinityfor FcγRIIA, when separately assayed for FcγR binding and/or effectorfunction in the context of the diabody molecule or other Fc containingmolecule (e.g., an immunoglobulin) comprising a wild-type Fc region asdescribed herein or known in the art; the combination of said “opposite”amino acid modification and selected IgG region then act to selectivelytemper or reduce a specific functionality in the diabody of theinvention relative to a diabody of the invention not comprising an Fcregion or comprising a wild-type Fc region of the same isotype.Alternatively, the invention encompasses variant Fc regions comprisingcombinations of amino acid modifications known in the art and selectedIgG regions that exhibit novel properties, which properties were notdetectable when said modifications and/or regions were independentlyassayed as described herein.

The functional characteristics of the multiple IgG isotypes, and domainsthereof, are well known in the art. The amino acid sequences of IgG1,IgG2, IgG3 and IgG4 are presented in FIGS. 1A-1B. Selection and/orcombinations of two or more domains from specific IgG isotypes for usein the methods of the invention may be based on any known parameter ofthe parent istoypes including affinity to FcγR (Table 7; Flesch andNeppert, 1999, J. Clin. Lab. Anal. 14:141-156; Chappel et al., 1993, J.Biol. Chem. 33:25124-25131; Chappel et al., 1991, Proc. Natl. Acad. Sci.USA 88:9036-9040, each of which is hereby incorporated by reference inits entirety). For example, use of regions or domains from IgG isotypesthat exhibit limited or no binding to FcγRIIB, e.g., IgG2 or IgG4, mayfind particular use where a diabody is desired to be engineered tomaximize binding to an activating receptor and minimize binding to aninhibitory receptor. Similarly, use of Fc regions or domains from IgGisotypes known to preferentially bind C1q or FcγRIIIA, e.g., IgG3(Brüggemann et al., 1987, J. Exp. Med 166:1351-1361), may be combinedwith Fc amino acid modifications of known in the art to enhance ADCC, toengineer a diabody molecule such that effector function activity, e.g.,complement activation or ADCC, is maximized. TABLE 7 Generalcharacteristics of IgG binding to FcγR, adapted from Flesch and Neppert,1999, J. Clin. Lab. Anal. 14: 141-156 Estimated Affinity for IgGReceptor (M⁻¹) Relative Affinity FcγRI 10⁸-10⁹ IgG3 > IgG1 >> IgG4no-binding: IgG2 FcγRIIA R^(131A) <10⁷ IgG3 > IgG1 no-binding: IgG2,IgG4 FcγRIIA H^(131A) <10⁷ IgG3 > IgG1 > IgG2 no-binding: IgG4FcγRIIB^(A) <10⁷ IgG3 > IgG1 > IgG4 no-binding: IgG2 FcγRIII <10⁷ IgG3 =IgG1 no-binding: IgG2, IgG4^(A)binds only complexed IgG5.3 Molecular Conjugates

The diabody molecules of the invention may be recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences.

Further, the diabody molecules of the invention (i.e., polypeptides) maybe conjugated to a therapeutic agent or a drug moiety that modifies agiven biological response. As an alternative to direct conjugation,owing to the multiple epitope binding sites on the multivalent, e.g.,tetravalent, diabody molecules of the invention, at least one bindingregion of the diabody may be designed to bind the therapeutic agent ordesired drug moiety without affecting diabody binding.

Therapeutic agents or drug moieties are not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin,gelonin, and pokeweed antiviral protein, a protein such as tumornecrosis factor, interferons including, but not limited to, α-interferon(IFN-α), β-interferon (IFN-β), nerve growth factor (NGF), plateletderived growth factor (PDGF), tissue plasminogen activator (TPA), anapoptotic agent (e.g., TNF-α, TNF-β, AIM I as disclosed in PCTPublication No. WO 97/33899), AIM II (see, PCT Publication No. WO97/34911), Fas ligand (Takahashi et al., J. Immunol., 6:1567-1574,1994), and VEGI (PCT Publication No. WO 99/23105), a thrombotic agent oran anti-angiogenic agent (e.g., angiostatin or endostatin), or abiological response modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”), macrophage colonystimulating factor, (“M-CSF”), or a growth factor (e.g., growth hormone(“GH”); proteases, or ribonucleases.

The diabody molecules of the invention (i.e., polypeptides) can be fusedto marker sequences, such as a peptide to facilitate purification. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., 1989, Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the hemagglutinin “HA” tag, which corresponds to an epitopederived from the influenza hemagglutinin protein (Wilson et al., Cell,37:767 1984) and the “flag” tag (Knappik et al., Biotechniques,17(4):754-761, 1994).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of molecules of the invention (e.g.,epitope binding sites with higher affinities and lower dissociationrates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721;5,834,252; and 5,837,458, and Patten et al., 1997, Curr. OpinionBiotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16:76; Hansson,et al., 1999, J. Mol. Biol. 287:265; and Lorenzo and Blasco, 1998,BioTechniques 24:308 (each of these patents and publications are herebyincorporated by reference in its entirety). The diabody molecules of theinvention, or the nucleic acids encoding the molecules of the invention,may be further altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. One or more portions of a polynucleotide encoding amolecule of the invention, may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

The present invention also encompasses diabody molecules of theinvention conjugated to or immunospecifically recognizing a diagnosticor therapeutic agent or any other molecule for which serum half-life isdesired to be increased/decreased and/or targeted to a particular subsetof cells. The molecules of the invention can be used diagnostically to,for example, monitor the development or progression of a disease,disorder or infection as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the molecules of the invention to a detectablesubstance or by the molecules immunospecifically recognizing thedetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to themolecules of the invention or indirectly, through an intermediate (suchas, for example, a linker known in the art) using techniques known inthe art, or the molecule may immunospecifically recognize the detectablesubstance: immunospecifically binding said substance. See, for example,U.S. Pat. No. 4,741,900 for metal ions which can be conjugated toantibodies for use as diagnostics according to the present invention.Such diagnosis and detection can be accomplished designing the moleculesto immunospecifically recognize the detectable substance or by couplingthe molecules of the invention to detectable substances including, butnot limited to, various enzymes, enzymes including, but not limited to,horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin; fluorescent materialssuch as, but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent material such as, but not limitedto, luminol; bioluminescent materials such as, but not limited to,luciferase, luciferin, and aequorin; radioactive material such as, butnot limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

The diabody molecules of the invention may immunospecifically recognizeor be conjugated to a therapeutic moiety such as a cytotoxin (e.g., acytostatic or cytocidal agent), a therapeutic agent or a radioactiveelement (e.g., alpha-emitters, gamma-emitters, etc.). Cytotoxins orcytotoxic agents include any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC), and anti-mitotic agents (e.g., vincristine andvinblastine).

Moreover, a diabody molecule of the invention can be conjugated to or bedesigned to immunospecifically recognize therapeutic moieties such as aradioactive materials or macrocyclic chelators useful for conjugatingradiometal ions (see above for examples of radioactive materials). Incertain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the polypeptide via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999, Bioconjug.Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943-50each of which is incorporated herein by reference in their entireties.

Techniques for conjugating such therapeutic moieties to polypeptides,including e.g., Fc domains, are well known; see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), 1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), 1985, pp. 475-506); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al., Immunol.Rev., 62:119-58, 1982.

The diabody molecule of the invention may be administered with orwithout a therapeutic moiety conjugated to it, administered alone, or incombination with cytotoxic factor(s) and/or cytokine(s) for use as atherapeutic treatment. Where administered alone, at least one epitope ofa multivalent, e.g., tetravalent, diabody molecule may be designed toimmunospecifically recognize a therapeutic agent, e.g., cytotoxicfactor(s) and/or cytokine(s), which may be administered concurrently orsubsequent to the molecule of the invention. In this manner, the diabodymolecule may specifically target the therapeutic agent in a mannersimilar to direct conjugation. Alternatively, a molecule of theinvention, can be conjugated to an antibody to form an antibodyheteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, whichis incorporated herein by reference in its entirety. Diabody moleculesof the invention may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

5.4 Characterization of Binding of Diabody Molecules

The diabody molecules of the present invention may be characterized in avariety of ways. In particular, molecules of the invention may beassayed for the ability to immunospecifically bind to an antigen, e.g.,FcRIIIA or FcRIIB, or, where the molecule comprises an Fc domain (orportion thereof) for the ability to exhibit Fc-FcγR interactions, i.e.specific binding of an Fc domain (or portion thereof) to an FcγR. Suchan assay may be performed in solution (e.g., Houghten, Bio/Techniques,13:412-421, 1992), on beads (Lam, Nature, 354:82-84, 1991, on chips(Fodor, Nature, 364:555-556, 1993), on bacteria (U.S. Pat. No.5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), on plasmids (Cull et al., Proc. Natl. Acad. Sci. USA,89:1865-1869, 1992) or on phage (Scott and Smith, Science, 249:386-390,1990; Devlin, Science, 249:404-406, 1990; Cwirla et al., Proc. Natl.Acad. Sci. USA, 87:6378-6382, 1990; and Felici, J. Mol. Biol.,222:301-310, 1991) (each of these references is incorporated byreference herein in its entirety). Molecules that have been identifiedto immunospecifically bind to an antigen, e.g., FcγRIIIA, can then beassayed for their specificity and affinity for the antigen.

Molecules of the invention that have been engineered to comprisemultiple epitope binding domains may be assayed for immunospecificbinding to one or more antigens (e.g., cancer antigen andcross-reactivity with other antigens (e.g., FcγR)) or, where themolecules comprise am Fc domain (or portion thereof) for Fc-FcγRinteractions by any method known in the art. Immunoassays which can beused to analyze immunospecific binding, cross-reactivity, and Fc-FcγRinteractions include, but are not limited to, competitive andnon-competitive assay systems using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays, to name but a few.Such assays are routine and well known in the art (see, e.g., Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York, which is incorporated by reference hereinin its entirety).

The binding affinity and the off-rate of antigen-binding domaininteraction or Fc-FcγR interaction can be determined by competitivebinding assays. One example of a competitive binding assay is aradioimmunoassay comprising the incubation of labeled antigen, such astetrameric FcγR (e.g., ³H or ¹²⁵I, see Section 5.4.1) with a molecule ofinterest (e.g., molecules of the present invention comprising multipleepitope binding domains in the presence of increasing amounts ofunlabeled epitope, such as tetrameric FcγR(see Section 5.4.1), and thedetection of the molecule bound to the labeled antigen. The affinity ofthe molecule of the present invention for an antigen and the bindingoff-rates can be determined from the saturation data by Scatchardanalysis.

The affinities and binding properties of the molecules of the inventionfor an antigen or FcγR may be initially determined using in vitro assays(biochemical or immunological based assays) known in the art forantigen-binding domain or Fc-FcγR, interactions, including but notlimited to ELISA assay, surface plasmon resonance assay,immunoprecipitation assays. Preferably, the binding properties of themolecules of the invention are also characterized by in vitro functionalassays for determining one or more FcγR mediator effector cellfunctions, as described in section 5.4.2. In most preferred embodiments,the molecules of the invention have similar binding properties in invivo models (such as those described and disclosed herein) as those inin vitro based assays. However, the present invention does not excludemolecules of the invention that do not exhibit the desired phenotype inin vitro based assays but do exhibit the desired phenotype in vivo.

In some embodiments, screening and identifying molecules comprisingmultiple epitope binding domains and, optionally, Fc domains (orportions thereof) are done functional based assays, preferably in a highthroughput manner. The functional based assays can be any assay known inthe art for characterizing one or more FcγR mediated effector cellfunctions such as those described herein in Sections 5.4.2 and 5.4.3.Non-limiting examples of effector cell functions that can be used inaccordance with the methods of the invention, include but are notlimited to, antibody-dependent cell mediated cytotoxicity (ADCC),antibody-dependent phagocytosis, phagocytosis, opsonization,opsonophagocytosis, cell binding, rosetting, C1q binding, and complementdependent cell mediated cytotoxicity.

In a preferred embodiment, BIAcore kinetic analysis is used to determinethe binding on and off rates of molecules of the present invention to anantigen or and FcγR. BIAcore kinetic analysis comprises analyzing thebinding and dissociation of an antigen or FcγR from chips withimmobilized molecules (e.g., molecules comprising epitope bindingdomains or Fc domains (or portions thereof), respectively) on theirsurface. BIAcore analysis is described in Section 5.4.3.

Preferably, fluorescence activated cell sorting (FACS), using any of thetechniques known to those skilled in the art, is used for immunologicalor functional based assay to characterize molecules of the invention.Flow sorters are capable of rapidly examining a large number ofindividual cells that have been bound, e.g., opsonized, by molecules ofthe invention (e.g., 10-100 million cells per hour) (Shapiro et al.,Practical Flow Cytometry, 1995). Additionally, specific parameters usedfor optimization of diabody behavior, include but are not limited to,antigen concentration (i.e., FcγR tetrameric complex, see Section5.4.1), kinetic competition time, or FACS stringency, each of which maybe varied in order to select for the diabody molecules comprisingmolecules of the invention which exhibit specific binding properties,e.g., concurrent binding to multiple epitopes. Flow cytometers forsorting and examining biological cells are well known in the art. Knownflow cytometers are described, for example, in U.S. Pat. Nos. 4,347,935;5,464,581; 5,483,469; 5,602,039; 5,643,796; and 6,211,477; the entirecontents of which are incorporated by reference herein. Other known flowcytometers are the FACS Vantage™ system manufactured by Becton Dickinsonand Company, and the COPAS™ system manufactured by Union Biometrica.

Characterization of target antigen binding affinity or Fc-FcγR bindingaffinity, and assessment of target antigen or FcγR density on a cellsurface may be made by methods well known in the art such as Scatchardanalysis or by the use of kits as per manufacturer's instructions, suchas Quantum™ Simply Cellular® (Bangs Laboratories, Inc., Fishers, IN).The one or more functional assays can be any assay known in the art forcharacterizing one or more FcγR mediated effector cell function as knownto one skilled in the art or described herein. In specific embodiments,the molecules of the invention comprising multiple epitope bindingdomains and, optionally, and Fc domain (or portion thereof) are assayedin an ELISA assay for binding to one or more target antigens or one ormore FcγRs, e.g., FcγRIIIA, FcγRIIA, FcγRIIA; followed by one or moreADCC assays. In some embodiments, the molecules of the invention areassayed further using a surface plasmon resonance-based assay, e.g.,BIAcore. Surface plasmon resonance-based assays are well known in theart, and are further discussed in Section 5.4.3, and exemplified herein,e.g., in Example 6.1.

In most preferred embodiments, the molecules of the invetion comprisingmultiple epitope binding domains and, optionally, and Fc domain (orportion thereof) is further characterized in an animal model forinteraction with a target antigen (e.g., an FcγR) or for Fc-FcγRinteraction. Where Fc-FcγR interactions are to be assessed, preferredanimal models for use in the methods of the invention are, for example,transgenic mice expressing human FcγRs, e.g., any mouse model describedin U.S. Pat. No. 5,877,397, and 6,676,927 which are incorporated hereinby reference in their entirety. Further transgenic mice for use in suchmethods include, but are not limited to, nude knockout FcγRIIIA micecarrying human FcγRIIIA; nude knockout FcγRIIIA mice carrying humanFcγRIIA; nude knockout FcγRIIIA mice carrying human FcγRIIB and humanFcγRIIIA; nude knockout FcγRIIIA mice carrying human FcγRIIB and humanFcγRIIA; nude knockout FcγRIIIA and FcγRIIA mice carrying human FcγRIIIAand FcγRIIA and nude knockout FcγRIIIA, FcγRIIA and FcγRIIB micecarrying human FcγRIIIA, FcγRIIA and FcγRIIB.

5.4.1 Binding Assays Comprising FcγR

Characterization of binding to FcγR by molecules comprising an Fc domain(or portion thereof) and/or comprising epitope binding domain specificfor an FcγR may be done using any FcγR, including but not limited topolymorphic variants of FcγR. In some embodiments, a polymorphic variantof FcγRIIIA is used, which contains a phenylalanine at position 158. Inother embodiments, characterization is done using a polymorphic variantof FcγRIIIA which contains a valine at position 158. FcγRIIIA 158Vdisplays a higher affinity for IgG1 than 158F and an increased ADCCactivity (see, e.g., Koene et al., 1997, Blood, 90:1109-14; Wu et al.,1997, J. Clin. Invest. 100: 1059-70, both of which are incorporatedherein by reference in their entireties); this residue in fact directlyinteracts with the lower hinge region of IgG1 as recently shown byIgG1-FcγRIIIA co-crystallization studies, see, e.g., Sonderman et al.,2000, Nature, 100: 1059-70, which is incorporated herein by reference inits entirety. Studies have shown that in some cases, therapeuticantibodies have improved efficacy in FcγRIIIA-158V homozygous patients.For example, humanized anti-CD20 monoclonal antibody Rituximab wastherapeutically more effective in FcγRIIIA158V homozygous patientscompared to FcγRIIIA 158F homozygous patients (See, e.g., Cartron etal., 2002 Blood, 99(3): 754-8). In other embodiments, therapeuticmolecules comprising this region may also be more effective on patientsheterozygous for FcγRIIIA-158V and FcγRIIIA-158F, and in patients withFcγRIIA-131H. Although not intending to be bound by a particularmechanism of action, selection of molecules of the invention withalternate allotypes may provide for variants that once engineered intotherapeutic diabodies will be clinically more efficacious for patientshomozygous for said allotype.

An FcγR binding assay was developed for determining the binding of themolecules of the invention to FcγR, and, in particular, for determiningbinding of Fc domains to FcγR. The assay allowed detection andquantitation of Fc-FcγR interactions, despite the inherently weakaffinity of the receptor for its ligand, e.g., in the micromolar rangefor FcγRIIB and FcγRIIIA. The method is described in detail inInternational Application WO04/063351 and U.S. Patent ApplicationPublications 2005/0037000 and 2005/0064514, each of which is herebyincorporated by reference in its entirety. Briefly, the method involvesthe formation of an FcγR complex that may be sued in any standardimmunoassay known in the art, e.g., FACS, ELISA, surface plasmonresonance, etc. Additionally, the FcγR complex has an improved avidityfor an Fc region, relative to an uncomplexed FcγR. According to theinvention, the preferred molecular complex is a tetrameric immunecomplex, comprising: (a) the soluble region of FcγR (e.g., the solubleregion of FcγRIIIA, FcγRIIA or FcγRIIB); (b) a biotinylated 15 aminoacid AVITAG sequence (AVITAG) operably linked to the C-terminus of thesoluble region of FcγR (e.g., the soluble region of FcγRIIIA, FcγRIIA orFcγRIIB); and (c) streptavidin-phycoerythrin (SA-PE); in a molar ratioto form a tetrameric FcγR complex (preferably in a 5:1 molar ratio). Thefusion protein is biotinylated enzymatically, using for example, the E.coli Bir A enzyme, a biotin ligase which specifically biotinylates alysine residue in the 15 amino acid AVITAG sequence. The biotinylatedsoluble FcγR proteins are then mixed with SA-PE in a 1X SA-PE:5Xbiotinylated soluble FcγR molar ratio to form a tetrameric FcγR complex.

Polypeptides comprising Fc regions have been shown to bind thetetrameric FcγR complexes with at least an 8-fold higher affinity thanthe monomeric uncomplexed FcγR. The binding of polypeptides comprisingFc regions to the tetrameric FcγR complexes may be determined usingstandard techniques known to those skilled in the art, such as forexample, fluorescence activated cell sorting (FACS), radioimmunoassays,ELISA assays, etc.

The invention encompasses the use of the immune complexes comprisingmolecules of the invention, and formed according to the methodsdescribed above, for determining the functionality of moleculescomprising an Fc region in cell-based or cell-free assays.

As a matter of convenience, the reagents may be provided in an assaykit, i.e., a packaged combination of reagents for assaying the abilityof molecules comprising an Fc regions to bind FcγR tetrameric complexes.Other forms of molecular complexes for use in determining Fc-FcγRinteractions are also contemplated for use in the methods of theinvention, e.g., fusion proteins formed as described in U.S. ProvisionalApplication 60/439,709, filed on Jan. 13, 2003; which is incorporatedherein by reference in its entirety.

5.4.2 Functional Assays of Molecules with Variant Heavy Chains

The invention encompasses characterization of the molecules of theinvention comprising multiple epitope binding domains and, optionally,Fc domains (or portions thereof) using assays known to those skilled inthe art for identifying the effector cell function of the molecules. Inparticular, the invention encompasses characterizing the molecules ofthe invention for FcγR-mediated effector cell function. Additionally,where at least one of the target antigens of the diabody molecule of theinvention is an FcγR, binding of the FcγR by the diabody molecule mayserve to activate FcγR-mediated pathways similar to those activated byFcγR-Fc binding. Thus, where at least one eptiope binding domain of thediabody molecule recognizes an FcγR, the diabody molecule may elicitFcγR-mediated effector cell function without containing an Fc domain (orportion thereof), or without concomitant Fc-FcγR binding. Examples ofeffector cell functions that can be assayed in accordance with theinvention, include but are not limited to, antibody-dependent cellmediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis,C1q binding, and complement dependent cell mediated cytotoxicity. Anycell-based or cell free assay known to those skilled in the art fordetermining effector cell function activity can be used (For effectorcell assays, see Perussia et al., 2000, Methods Mol. Biol. 121: 179-92;Baggiolini et al., 1998 Experientia, 44(10): 841-8; Lehmann et al., 2000J. Immunol. Methods, 243(1-2): 229-42; Brown E J. 1994, Methods CellBiol., 45: 147-64; Munn et al., 1990 J. Exp. Med., 172: 231-237,Abdul-Majid et al., 2002 Scand. J. Immunol. 55: 70-81; Ding et al.,1998, Immunity 8:403-411, each of which is incorporated by referenceherein in its entirety).

In one embodiment, the molecules of the invention can be assayed forFcγR-mediated phagocytosis in human monocytes. Alternatively, theFcγR-mediated phagocytosis of the molecules of the invention may beassayed in other phagocytes, e.g., neutrophils (polymorphonuclearleuckocytes; PMN); human peripheral blood monocytes, monocyte-derivedmacrophages, which can be obtained using standard procedures known tothose skilled in the art (e.g., see Brown E J. 1994, Methods Cell Biol.,45: 147-164). In one embodiment, the function of the molecules of theinvention is characterized by measuring the ability of THP-1 cells tophagocytose fluoresceinated IgG-opsonized sheep red blood cells (SRBC)by methods previously described (Tridandapani et al., 2000, J. Biol.Chem. 275: 20480-7).

Another exemplary assay for determining the phagocytosis of themolecules of the invention is an antibody-dependent opsonophagocytosisassay (ADCP) which can comprise the following: coating a targetbioparticle such as Escherichia coli-labeled FITC (Molecular Probes) orStaphylococcus aureus-FITC with (i) wild-type 4-4-20 antibody, anantibody to fluorescein (See Bedzyk et al., 1989, J. Biol. Chem, 264(3):1565-1569, which is incorporated herein by reference in its entirety),as the control antibody for FcγR-dependent ADCP; or (ii) 4-4-20 antibodyharboring the D265A mutation that knocks out binding to FcγRIII, as abackground control for FcγR-dependent ADCP (iii) a diabody comprisingthe epitope binding domain of 4-4-20 and an Fc domain and/or an epitopebinding domain specific for FcγRIII; and forming the opsonized particle;adding any of the opsonized particles described (i-iii) to THP-1effector cells (a monocytic cell line available from ATCC) at a 1:1,10:1, 30:1, 60:1, 75:1 or a 100: 1 ratio to allow FcγR-mediatedphagocytosis to occur; preferably incubating the cells and E.coli-FITC/antibody at 37° C. for 1.5 hour; adding trypan blue afterincubation (preferably at room temperature for 2-3 min.) to the cells toquench the fluoroscence of the bacteria that are adhered to the outsideof the cell surface without being internalized; transferring cells intoa FACS buffer (e.g., 0.1%, BSA in PBS, 0.1%, sodium azide), analyzingthe fluorescence of the THP1 cells using FACS (e.g., BD FACS Calibur).Preferably, the THP-1 cells used in the assay are analyzed by FACS forexpression of FcγR on the cell surface. THP-1 cells express both CD32Aand CD64. CD64 is a high affinity FcγR that is blocked in conducting theADCP assay in accordance with the methods of the invention. The THP-1cells are preferably blocked with 100 μg/mL soluble IgG1 or 10% humanserum. To analyze the extent of ADCP, the gate is preferably set onTHP-1 cells and median fluorescence intensity is measured. The ADCPactivity for individual mutants is calculated and reported as anormalized value to the wild type chMab 4-4-20 obtained. The opsonizedparticles are added to THP-1 cells such that the ratio of the opsonizedparticles to THP-1 cells is 30:1 or 60:1. In most preferred embodiments,the ADCP assay is conducted with controls, such as E. coli-FITC inmedium, E. coli-FITC and THP-1 cells (to serve as FcγR-independent ADCPactivity), E. coli-FITC, THP-1 cells and wild-type 4-4-20 antibody (toserve as FcγR-dependent ADCP activity), E coli-FITC, THP-1 cells, 4-4-20D265A (to serve as the background control for FcγR-dependent ADCPactivity).

In another embodiment, the molecules of the invention can be assayed forFcγR-mediated ADCC activity in effector cells, e.g., natural killercells, using any of the standard methods known to those skilled in theart (See e.g., Perussia et al., 2000, Methods Mol. Biol. 121: 179-92;Weng et al., 2003, J. Clin. Oncol. 21:3940-3947; Ding et al., Immunity,1998, 8:403-11). An exemplary assay for determining ADCC activity of themolecules of the invention is based on a ⁵¹Cr release assay comprisingof: labeling target cells with [⁵¹Cr]Na₂CrO₄ (this cell-membranepermeable molecule is commonly used for labeling since it bindscytoplasmic proteins and although spontaneously released from the cellswith slow kinetics, it is released massively following target cellnecrosis); opsonizing the target cells with the molecules of theinvention comprising variant heavy chains; combining the opsonizedradiolabeled target cells with effector cells in a microtitre plate atan appropriate ratio of target cells to effector cells; incubating themixture of cells for 16-18 hours at 37° C.; collecting supernatants; andanalyzing radioactivity. The cytotoxicity of the molecules of theinvention can then be determined, for example using the followingformula: % lysis=(experimental cpm−target leak cpm)/(detergent lysiscpm−target leak cpm)×100%. Alternatively, % lysis=(ADCC−AICC)/(maximumrelease−spontaneous release). Specific lysis can be calculated using theformula: specific lysis=% lysis with the molecules of the invention −%lysis in the absence of the molecules of the invention. A graph can begenerated by varying either the target: effector cell ratio or antibodyconcentration.

Preferably, the effector cells used in the ADCC assays of the inventionare peripheral blood mononuclear cells (PBMC) that are preferablypurified from normal human blood, using standard methods known to oneskilled in the art, e.g., using Ficoll-Paque density gradientcentrifugation. Preferred effector cells for use in the methods of theinvention express different FcγR activating receptors. The inventionencompasses, effector cells, THP-1, expressing FcγRI, FcγRIIA andFcγRIIB, and monocyte derived primary macrophages derived from wholehuman blood expressing both FcγRIIIA and FcγRIIB, to determine if heavychain antibody mutants show increased ADCC activity and phagocytosisrelative to wild type IgG1 antibodies.

The human monocyte cell line, THP-1, activates phagocytosis throughexpression of the high affinity receptor FcγRI and the low affinityreceptor FcγRIIA (Fleit et al., 1991, J. Leuk. Biol. 49: 556). THP-1cells do not constitutively express FcγRIIA or FcγRIIB. Stimulation ofthese cells with cytokines effects the FcR expression pattern (Pricop etal., 2000 J. Immunol. 166: 531-7). Growth of THP-1 cells in the presenceof the cytokine IL4 induces FcγRIIB expression and causes a reduction inFcγRIIA and FcγRI expression. FcγRIIB expression can also be enhanced byincreased cell density (Tridandapani et al., 2002, J. Biol Chem. 277:5082-9). In contrast, it has been reported that IFNγ can lead toexpression of FcγRIIIA (Pearse et al., 1993 PNAS USA 90: 4314-8). Thepresence or absence of receptors on the cell surface can be determinedby FACS using common methods known to one skilled in the art. Cytokineinduced expression of FcγR on the cell surface provides a system to testboth activation and inhibition in the presence of FcγRIIB. If THP-1cells are unable to express the FcγRIIB the invention also encompassesanother human monocyte cell line, U937. These cells have been shown toterminally differentiate into macrophages in the presence of IFNγ andTNF (Koren et al., 1979, Nature 279: 328-331).

FcγR dependent tumor cell killing is mediated by macrophage and NK cellsin mouse tumor models (Clynes et al., 1998, PNAS USA 95: 652-656). Theinvention encompasses the use of elutriated monocytes from donors aseffector cells to analyze the efficiency Fc mutants to trigger cellcytotoxicity of target cells in both phagocytosis and ADCC assays.Expression patterns of FcγRI, FcγRIIIA, and FcγRIIB are affected bydifferent growth conditions. FcγR expression from frozen elutriatedmonocytes, fresh elutriated monocytes, monocytes maintained in 10% FBS,and monocytes cultured in FBS+GM-CSF and or in human serum may bedetermined using common methods known to those skilled in the art. Forexample, cells can be stained with FcγR specific antibodies and analyzedby FACS to determine FcR profiles. Conditions that best mimic macrophagein vivo FcγR expression is then used for the methods of the invention.

In some embodiments, the invention encompasses the use of mouse cellsespecially when human cells with the right FcγR profiles are unable tobe obtained. In some embodiments, the invention encompasses the mousemacrophage cell line RAW264.7(ATCC) which can be transfected with humanFcγRIIIA and stable transfectants isolated using methods known in theart, see, e.g., Ralph et al., J. Immunol. 119: 950-4). Transfectants canbe quantitated for FcγRIIIA expression by FACS analysis using routineexperimentation and high expressors can be used in the ADCC assays ofthe invention. In other embodiments, the invention encompasses isolationof spleen peritoneal macrophage expressing human FcγR from knockouttransgenic mice such as those disclosed herein.

Lymphocytes may be harvested from peripheral blood of donors (PBM) usinga Ficoll-Paque gradient (Pharmacia). Within the isolated mononuclearpopulation of cells the majority of the ADCC activity occurs via thenatural killer cells (NK) containing FcγRIIIA but not FcγRIIB on theirsurface. Results with these cells indicate the efficacy of the mutantson triggering NK cell ADCC and establish the reagents to test withelutriated monocytes.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Tremp et al., 1976, Cancer Res.33-41); B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Rajicells with ATCC accession number CCL-86 (see, e.g., Epstein et al.,1965, J. Natl. Cancer Inst. 34: 231-240), and Daudi cells with ATCCaccession number CCL-213 (see, e.g., Klein et al., 1968, Cancer Res. 28:1300-10). The target cells must be recognized by the antigen bindingsite of the diabody molecule to be assayed.

The ADCC assay is based on the ability of NK cells to mediate cell deathvia an apoptotic pathway. NK cells mediate cell death in part byFcγRIIIA's recognition of an IgG Fc domain bound to an antigen on a cellsurface. The ADCC assays used in accordance with the methods of theinvention may be radioactive based assays or fluorescence based assays.The ADCC assay used to characterize the molecules of the inventioncomprising variant Fc regions comprises labeling target cells, e.g.,SK-BR-3, MCF-7, OVCAR3, Raji, Daudi cells, opsonizing target cells withan antibody that recognizes a cell surface receptor on the target cellvia its antigen binding site; combining the labeled opsonized targetcells and the effector cells at an appropriate ratio, which can bedetermined by routine experimentation; harvesting the cells; detectingthe label in the supernatant of the lysed target cells, using anappropriate detection scheme based on the label used. The target cellsmay be labeled either with a radioactive label or a fluorescent label,using standard methods known in the art. For example the labels include,but are not limited to, [⁵¹Cr]Na₂CrO₄; and the acetoxymethyl ester ofthe fluorescence enhancing ligand,2,2′:6′,2″-terpyridine-6-6″-dicarboxylate (TDA).

In a specific preferred embodiment, a time resolved fluorimetric assayis used for measuring ADCC activity against target cells that have beenlabeled with the acetoxymethyl ester of the fluorescence enhancingligand, 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate (TDA). Suchfluorimetric assays are known in the art, e.g., see, Blomberg et al.,1996, Journal of Immunological Methods, 193: 199-206; which isincorporated herein by reference in its entirety. Briefly, target cellsare labeled with the membrane permeable acetoxymethyl diester of TDA(bis(acetoxymethyl) 2,2′:6′,2″-terpyridine-6-6″-dicarboxylate, (BATDA),which rapidly diffuses across the cell membrane of viable cells.Intracellular esterases split off the ester groups and the regeneratedmembrane impermeable TDA molecule is trapped inside the cell. Afterincubation of effector and target cells, e.g., for at least two hours,up to 3.5 hours, at 37° C., under 5% CO₂, the TDA released from thelysed target cells is chelated with Eu3+ and the fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallace).

In another specific embodiment, the ADCC assay used to characterize themolecules of the invention comprising multiple epitope binding sitesand, optionally, an Fc domain (or portion thereof) comprises thefollowing steps: Preferably 4-5×10⁶ target cells (e.g., SK-BR-3, MCF-7,OVCAR3, Raji cells) are labeled with bis(acetoxymethyl)2,2′:6′,2″-terpyridine-t-6″-dicarboxylate (DELFIA BATDA Reagent, PerkinElmer/Wallac). For optimal labeling efficiency, the number of targetcells used in the ADCC assay should preferably not exceed 5×10⁶. BATDAreagent is added to the cells and the mixture is incubated at 37° C.preferably under 5% CO₂, for at least 30 minutes. The cells are thenwashed with a physiological buffer, e.g., PBS with 0.125 mMsulfinpyrazole, and media containing 0.125 mM sulfinpyrazole. Thelabeled target cells are then opsonized (coated) with a molecule of theinvention comprising an epitope binding domain specific for FcγRIIA and,optionally, an Fc domain (or portion thereof). In preferred embodiments,the molecule used in the ADCC assay is also specific for a cell surfacereceptor, a tumor antigen, or a cancer antigen. The diabody molecule ofthe invetion may specifically bind any cancer or tumor antigen, such asthose listed in section 5.6.1. The target cells in the ADCC assay arechosen according to the epitope binding sites engineered into thediabody of the invention, such that the diabody binds a cell surfacereceptor of the target cell specifically.

Target cells are added to effector cells, e.g., PBMC, to produceeffector:target ratios of approximately 1:1, 10:1, 30:1, 50:1, 75:1, or100:1. The effector and target cells are incubated for at least twohours, up to 3.5 hours, at 37° C., under 5% CO₂. Cell supernatants areharvested and added to an acidic europium solution (e.g., DELFIAEuropium Solution, Perkin Elmer/Wallac). The fluorescence of theEuropium-TDA chelates formed is quantitated in a time-resolvedfluorometer (e.g., Victor 1420, Perkin Elmer/Wallac). Maximal release(MR) and spontaneous release (SR) are determined by incubation of targetcells with 1% TX-100 and media alone, respectively. Antibody independentcellular cytotoxicity (AICC) is measured by incubation of target andeffector cells in the absence of a test molecule, e.g., diabody of theinvention. Each assay is preferably performed in triplicate. The meanpercentage specific lysis is calculated as: Experimental release(ADCC)-AICC)/(MR-SR)×100.

The invention encompasses assays known in the art, and exemplifiedherein, to characterize the binding of C1q and mediation of complementdependent cytotoxicity (CDC) by molecules of the invention comprising Fcdomains (or portions thereof). To determine C1q binding, a C1q bindingELISA may be performed. An exemplary assay may comprise the following:assay plates may be coated overnight at 4C with polypeptide comprising amolecule of the invention or starting polypeptide (control) in coatingbuffer. The plates may then be washed and blocked. Following washing, analiquot of human C1q may be added to each well and incubated for 2 hrsat room temperature. Following a further wash, 100 uL of a sheepanti-complement C1q peroxidase conjugated antibody may be added to eachwell and incubated for 1 hour at room temperature. The plate may againbe washed with wash buffer and 100 ul of substrate buffer containing OPD(O-phenylenediamine dihydrochloride (Sigma)) may be added to each well.The oxidation reaction, observed by the appearance of a yellow color,may be allowed to proceed for 30 minutes and stopped by the addition of100 ul of 4.5 NH2 SO4. The absorbance may then read at (492-405) nm.

To assess complement activation, a complement dependent cytotoxicity(CDC) assay may be performed, e.g as described in Gazzano-Santoro etal., J. Immunol. Methods 202:163 (1996), which is incorporated herein byreference in its entirety. Briefly, various concentrations of themolecule comprising a (variant) Fc domain (or portion thereof) and humancomplement may be diluted with buffer. Cells which express the antigento which the diabody molecule binds may be diluted to a density of about1×10⁶ cells/ml. Mixtures of the diabody molecules comprising a (variant)Fc domain (or portion thereof), diluted human complement and cellsexpressing the antigen may be added to a flat bottom tissue culture 96well plate and allowed to incubate for 2 hrs at 37 C. and 5% CO2 tofacilitate complement mediated cell lysis. 50 uL of alamar blue (AccumedInternational) may then be added to each well and incubated overnight at37 C. The absorbance is measured using a 96-well fluorometer withexcitation at 530 nm and emission at 590 nm. The results may beexpressed in relative fluorescence units (RFU). The sampleconcentrations may be computed from a standard curve and the percentactivity as compared to nonvariant molecule, i.e., a molecule notcomprising an Fc domain or comprising a non-variant Fc domain, isreported for the variant of interest.

5.4.3 Other Assays

The molecules of the invention comprising multiple epitope bindingdomain and, optionally, an Fc domain may be assayed using any surfaceplasmon resonance based assays known in the art for characterizing thekinetic parameters of an antigen-binding domain or Fc-FcγR binding. AnySPR instrument commercially available including, but not limited to,BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); IAsysinstruments available from Affinity Sensors (Franklin, Mass.); IBISsystem available from Windsor Scientific Limited (Berks, UK), SPR-CELLIAsystems available from Nippon Laser and Electronics Lab (Hokkaido,Japan), and SPR Detector Spreeta available from Texas Instruments(Dallas, Tex.) can be used in the instant invention. For a review ofSPR-based technology see Mullet et al., 2000, Methods 22: 77-91; Dong etal., 2002, Review in Mol. Biotech., 82: 303-23; Fivash et al., 1998,Current Opinion in Biotechnology 9: 97-101; Rich et al., 2000, CurrentOpinion in Biotechnology 11: 54-61; all of which are incorporated hereinby reference in their entirety. Additionally, any of the SPR instrumentsand SPR based methods for measuring protein-protein interactionsdescribed in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215;6,268,125 are contemplated in the methods of the invention, all of whichare incorporated herein by reference in their entirety.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occur is the sensor chip, which is at the heart ofthe SPR technology; it consists of a glass surface coated with a thinlayer of gold and forms the basis for a range of specialized surfacesdesigned to optimize the binding of a molecule to the surface. A varietyof sensor chips are commercially available especially from the companieslisted supra, all of which may be used in the methods of the invention.Examples of sensor chips include those available from BIAcore AB, Inc.,e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention maybe immobilized onto the surface of a sensor chip using any of theimmobilization methods and chemistries known in the art, including butnot limited to, direct covalent coupling via amine groups, directcovalent coupling via sulfhydryl groups, biotin attachment to avidincoated surface, aldehyde coupling to carbohydrate groups, and attachmentthrough the histidine tag with NTA chips.

In some embodiments, the kinetic parameters of the binding of moleculesof the invention comprising multiple epitope binding sites and,optionally, and Fc domain, to an antigen or an FcγR may be determinedusing a BIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc.,Piscataway, N.J.). As discussed supra, see section 5.4.1, any FcγR canbe used to assess the binding of the molecules of the invention eitherwhere at least one epitope binding site of the diabody moleculeimmunospecifically recognizes an FcγR, and/or where the diabody moleculecomprises an Fc domain (or portion thereof). In a specific embodimentthe FcγR is FcγRIIIA, preferably a soluble monomeric FcγRIIIA. Forexample, in one embodiment, the soluble monomeric FcγRIIIA is theextracellular region of FcγRIIIA joined to the linker-AVITAG sequence(see, U.S. Provisional Application No. 60/439,498, filed on Jan. 9, 2003(Attorney Docket No. 11183-004-888 and U.S. Provisional Application No.60/456,041 filed on Mar. 19, 2003, which are incorporated herein byreference in their entireties). In another specific embodiment, the FcγRis FcγRIIB, preferably a soluble dimeric FcγRIIB. For example in oneembodiment, the soluble dimeric FcγRIIB protein is prepared inaccordance with the methodology described in U.S. Provisionalapplication No. 60/439,709 filed on Jan. 13, 2003, which is incorporatedherein by reference in its entirety.

For all immunological assays, FcγR recognition/binding by a molecule ofthe invention may be effected by multiple domains: in certainembodiments, molecules of the invention immunospecifically recognize anFcγR via one of the multiple epitope binding domains; in yet otherembodiments, where the molecule of the invetion comprises an Fc domain(or portion thereof), the diabody molecule may immunospecificallyrecognize an FcγR via Fc-FcγR interactions; in yet further embodiments,where a molecule of the invetion comprises both an Fc domain (or portionthereof) and an epitope binding site that immunospecifically recognizesan FcγR, the diabody molecule may recognize an FcγR via one or both ofan epitope binding domain and the Fc domain (or portion thereof). Anexemplary assay for determining the kinetic parameters of a moleculecomprising multiple epitope binding domains and, optionally, and Fcdomain (or portion thereof) to an antigen and/or an FcγR using a BIAcoreinstrument comprises the following: a first antigen is immobilized onone of the four flow cells of a sensor chip surface, preferably throughamine coupling chemistry such that about 5000 response units (RU) ofsaid first antigen is immobilized on the surface. Once a suitablesurface is prepared, molecules of the invention that immunospecificallyrecognize said first antigen are passed over the surface, preferably byone minute injections of a 20 μg/mL solution at a 5 μL/mL flow rate.Levels of molecules of the invention bound to the surface at this stagetypically ranges between 400 and 700 RU. Next, dilution series of asecond antigen (e.g., FcγR) or FcγR receptor in HBS-P buffer (20 mMHEPES, 150 mM NaCl, 3 mM EDTA, pH 7.5) are injected onto the surface at100 μL/min Regeneration of molecules between different second antigen orreceptor dilutions is carried out preferably by single 5 secondinjections of 100 mM NaHCO₃ pH 9.4; 3M NaCl. Any regeneration techniqueknown in the art is contemplated in the method of the invention.

Once an entire data set is collected, the resulting binding curves areglobally fitted using computer algorithms supplied by the SPR instrumentmanufacturer, e.g., BIAcore, Inc. (Piscataway, N.J.). These algorithmscalculate both the K_(on) and K_(off) from which the apparentequilibrium binding constant, K_(d) is deduced as the ratio of the tworate constants (i.e., K_(off)/K_(on)). More detailed treatments of howthe individual rate constants are derived can be found in theBIAevaluaion Software Handbook (BIAcore, Inc., Piscataway, N.J.). Theanalysis of the generated data may be done using any method known in theart. For a review of the various methods of interpretation of thekinetic data generated see Myszka, 1997, Current Opinion inBiotechnology 8: 50-7; Fisher et al., 1994, Current Opinion inBiotechnology 5: 389-95; O'Shannessy, 1994, Current Opinion inBiotechnology, 5:65-71; Chaiken et al., 1992, Analytical Biochemistry,201: 197-210; Morton et al., 1995, Analytical Biochemistry 227: 176-85;O'Shannessy et al., 1996, Analytical Biochemistry 236: 275-83; all ofwhich are incorporated herein by reference in their entirety.

In preferred embodiments, the kinetic parameters determined using an SPRanalysis, e.g., BIAcore, may be used as a predictive measure of how amolecule of the invention will function in a functional assay, e.g.,ADCC. An exemplary method for predicting the efficacy of a molecule ofthe invention based on kinetic parameters obtained from an SPR analysismay comprise the following: determining the K_(off) values for bindingof a molecule of the invention to FcγRIIIA and FcγRIIB (via an epitopebinding domain and/or an Fc domain (or portion thereof)); plotting (1)K_(off)(wt)/K_(off) (mut) for FcγRIIIA; (2) K_(off) (mut)/K_(off) (wt)for FcγRIIB against the ADCC data. Numbers higher than one show adecreased dissociation rate for FcγRIIIA and an increased dissociationrate for FcγRIIB relative to wild type; and possess and enhanced ADCCfunction.

5.5 Methods of Producing Diabody Molecules of the Invention

The diabody molecules of the present invention can be produced using avariety of methods well known in the art, including de novo proteinsynthesis and recombinant expression of nucleic acids encoding thebinding proteins. The desired nucleic acid sequences can be produced byrecombinant methods (e.g., PCR mutagenesis of an earlier preparedvariant of the desired polynucleotide) or by solid-phase DNA synthesis.Usually recombinant expression methods are used. In one aspect, theinvention provides a polynucleotide that comprises a sequence encoding aCD16A VH and/or VL; in another aspect, the invention provides apolynucleotide that comprises a sequence encoding a CD32B VH and/or VL.Because of the degeneracy of the genetic code, a variety of nucleic acidsequences encode each immunoglobulin amino acid sequence, and thepresent invention includes all nucleic acids encoding the bindingproteins described herein.

5.5.1 Polynucleotides Encoding Molecules of the Invention.

The present invention also includes polynucleotides that encode themolecules of the invention, including the polypeptides and antibodies.The polynucleotides encoding the molecules of the invention may beobtained, and the nucleotide sequence of the polynucleotides determined,by any method known in the art.

Once the nucleotide sequence of the molecules that are identified by themethods of the invention is determined, the nucleotide sequence may bemanipulated using methods well known in the art, e.g., recombinant DNAtechniques, site directed mutagenesis, PCR, etc. (see, for example, thetechniques described in Sambrook et al., 2001, Molecular Cloning, ALaboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.; and Ausubel et al., eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY, which are both incorporated byreference herein in their entireties), to generate, for example,antibodies having a different amino acid sequence, for example bygenerating amino acid substitutions, deletions, and/or insertions.

In one embodiment, human libraries or any other libraries available inthe art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the molecules of the invention.

5.5.2 Recombinant Expression of Molecules of the Invention

Once a nucleic acid sequence encoding molecules of the invention (i.e.,antibodies) has been obtained, the vector for the production of themolecules may be produced by recombinant DNA technology using techniqueswell known in the art. Methods which are well known to those skilled inthe art can be used to construct expression vectors containing thecoding sequences for the molecules of the invention and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, for example, thetechniques described in Sambrook et al., 1990, Molecular Cloning, ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. and Ausubel et al. eds., 1998, Current Protocols inMolecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of a moleculeidentified by the methods of the invention can be transferred to a hostcell by conventional techniques (e.g., electroporation, liposomaltransfection, and calcium phosphate precipitation) and the transfectedcells are then cultured by conventional techniques to produce themolecules of the invention. In specific embodiments, the expression ofthe molecules of the invention is regulated by a constitutive, aninducible or a tissue, specific promoter.

The host cells used to express the molecules identified by the methodsof the invention may be either bacterial cells such as Escherichia coli,or, preferably, eukaryotic cells, especially for the expression of wholerecombinant immunoglobulin molecule. In particular, mammalian cells suchas Chinese hamster ovary cells (CHO), in conjunction with a vector suchas the major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for immunoglobulins(Foecking et al., 1998, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

A variety of host-expression vector systems may be utilized to expressthe molecules identified by the methods of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof the molecules of the invention may be produced and subsequentlypurified, but also represent cells which may, when transformed ortransfected with the appropriate nucleotide coding sequences, expressthe molecules of the invention in situ. These include, but are notlimited to, microorganisms such as bacteria (e.g., E. coli and B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing coding sequences for themolecules identified by the methods of the invention; yeast (e.g.,Saccharomyces Pichia) transformed with recombinant yeast expressionvectors containing sequences encoding the molecules identified by themethods of the invention; insect cell systems infected with recombinantvirus expression vectors (e.g., baclovirus) containing the sequencesencoding the molecules identified by the methods of the invention; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing sequences encoding the molecules identified by themethods of the invention; or mammalian cell systems (e.g., COS, CHO,BHK, 293, 293T, 3T3 cells, lymphotic cells (see U.S. Pat. No.5,807,715), Per C.6 cells (human retinal cells developed by Crucell)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the moleculebeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibodycoding sequence may be ligated individually into the vector in framewith the lac Z coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (e.g., the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (e.g., thepolyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods inEnzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. For example, in certainembodiments, the polypeptides comprising a diabody molecule of theinvention may be expressed as a single gene product (e.g., as a singlepolypeptide chain, i.e., as a polyprotein precursor), requiringproteolytic cleavage by native or recombinant cellular mechanisms toform the separate polypeptides of the diabody molecules of theinvention. The invention thus encompasses engineering a nucleic acidsequence to encode a polyprotein precursor molecule comprising thepolypeptides of the invention, which includes coding sequences capableof directing post translational cleavage of said polyprotein precursor.Post-translational cleavage of the polyprotein precursor results in thepolypeptides of the invention. The post translational cleavage of theprecursor molecule comprising the polypeptides of the invention mayoccur in vivo (i.e., within the host cell by native or recombinant cellsystems/mechanisms, e.g. furin cleavage at an appropriate site) or mayoccur in vitro (e.g incubation of said polypeptide chain in acomposition comprising proteases or peptidases of known activity and/orin a composition comprising conditions or reagents known to foster thedesired proteolytic action). Purification and modification ofrecombinant proteins is well known in the art such that the design ofthe polyprotein precursor could include a number of embodiments readilyappreciated by a skilled worker. Any known proteases or peptidases knownin the art can be used for the described modification of the precursormolecule, e.g., thrombin (which recognizes the amino acid sequenceLVPRˆGS (SEQ ID NO:91)), or factor Xa (which recognizes the amino acidsequence I(E/D)GRˆ (SEQ ID NO:92) (Nagani et al., 1985, PNAS USA82:7252-7255, and reviewed in Jenny et al., 2003, Protein Expr. Purif.31:1-11, each of which is incorporated by reference herein in itsentirety)), enterokinase (which recognizes the amino acid sequenceDDDDKˆ (SEQ ID NO:93) (Collins-Racie et al., 1995, Biotechnol.13:982-987 hereby incorporated by reference herein in its entirety)),furin (which recognizes the amino acid sequence RXXRˆ, with a preferencefor RX(K/R)Rˆ (SEQ ID NO:94 and SEQ ID NO:95, respectively) (additionalR at P6 position appears to enhance cleavage)), and AcTEV (whichrecognizes the amino acid sequence ENLYFQˆG (SEQ ID NO:96) (Parks etal., 1994, Anal. Biochem. 216:413 hereby incorporated by referenceherein in its entirety)) and the Foot and Mouth Disease Virus ProteaseC3. See for example, section 6.4, supra.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Suchmammalian host cells include but are not limited to CHO, VERY, BHK,Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20 andT47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably express anantibody of the invention may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express theantibodies of the invention. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatinteract directly or indirectly with the molecules of the invention.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48: 202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, 3:87-95; Tolstoshev,1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem.62:191-217; May, 1993, TIB TECH 11(5):155-215). Methods commonly knownin the art of recombinant DNA technology which can be used are describedin Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology,John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, NY; and in Chapters 12 and 13,Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, JohnWiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1;and hygro, which confers resistance to hygromycin (Santerre et al.,1984, Gene 30:147).

The expression levels of a molecule of the invention can be increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987). When a marker in the vector system expressing an antibodyis amplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of apolypeptide of the diabody molecule, production of the polypeptide willalso increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding the first polypeptide of thediabody molecule and the second vector encoding the second polypeptideof the diabody molecule. The two vectors may contain identicalselectable markers which enable equal expression of both polypeptides.Alternatively, a single vector may be used which encodes bothpolypeptides. The coding sequences for the polypeptides of the moleculesof the invention may comprise cDNA or genomic DNA.

Once a molecule of the invention (i.e., diabodies) has beenrecombinantly expressed, it may be purified by any method known in theart for purification of polypeptides, polyproteins or diabodies (e.g.,analogous to antibody purification schemes based on antigen selectivity)for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen (optionally afterProtein A selection where the diabody molecule comprises an Fc domain(or portion thereof)), and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of polypeptides, polyproteins ordiabodies.

5.6 Prophylactic and Therapeutic Methods

The molecules of the invention are particularly useful for the treatmentand/or prevention of a disease, disorder or infection where an effectorcell function (e.g., ADCC) mediated by FcγR is desired (e.g., cancer,infectious disease). As discussed supra, the diabodies of the invetionmay exhibit antibody-like functionality in eliciting effector functionalthough the diabody molecule does not comprise and Fc domain. Bycomprising at least one epitope binding domain that immunospecificallyrecognizes an FcγR, the diabody molecule may exibit FcγR binding andactivity analogous to Fc-FcγR interactions. For example, molecules ofthe invention may bind a cell surface antigen and an FcγR (e.g.,FcγRIIIA) on an immune effector cell (e.g., NK cell), stimulating aneffector function (e.g., ADCC, CDC, phagocytosis, opsonization, etc.)against said cell.

In other embodiments, the diabody molecule of the invention comprises anFc domain (or portion thereof). In such embodiments, the Fc domain mayfurther comprise at least one amino acid modification relative to awild-type Fc domain (or portion thereof) and/or may comprise domainsfrom one or more IgG isotypes (e.g., IgG1, IgG2, IgG3 or IgG4).Molecules of the invetion comprising variant Fc domains may exhibitconferred or altered phenotypes relative to molecules comprising thewild type Fc domain such as an altered or conferred effector functionactivity (e.g., as assayed in an NK dependent or macrophage dependentassay). In said embodiments, molecules of the invention with conferredor altered effector function activity are useful for the treatmentand/or prevention of a disease, disorder or infection where an enhancedefficacy of effector function activity is desired. In certainembodiments, the diabody molecules of the invention comprising an Fcdomain (or portion thereof) mediate complement dependent cascade. Fcdomain variants identified as altering effector function are disclosedin International Application WO04/063351, U.S. Patent ApplicationPublications 2005/0037000 and 2005/0064514, U.S. ProvisionalApplications 60/626,510, filed Nov. 10, 2004, 60/636,663, filed Dec. 15,2004, and 60/781,564, filed Mar. 10, 2006, and U.S. patent applicationsSer. No. 11/271,140, filed Nov. 10, 2005, and Ser. No. 11/305,787, filedDec. 15, 2005, concurrent applications of the Inventors, each of whichis incorporated by reference in its entirety.

The invention encompasses methods and compositions for treatment,prevention or management of a cancer in a subject, comprisingadministering to the subject a therapeutically effective amount of oneor more molecules comprising one or more epitope binding sites, andoptionally, an Fc domain (or portion thereof) engineered in accordancewith the invention, which molecule further binds a cancer antigen.Molecules of the invention are particularly useful for the prevention,inhibition, reduction of growth or regression of primary tumors,metastasis of cancer cells, and infectious diseases. Although notintending to be bound by a particular mechanism of action, molecules ofthe invention mediate effector function resulting in tumor clearance,tumor reduction or a combination thereof. In alternate embodiments, thediabodies of the invention mediate therapeutic activity by cross-linkingof cell surface antigens and/or receptors and enhanced apoptosis ornegative growth regulatory signaling.

Although not intending to be bound by a particular mechanism of action,the diabody molecules of the invention exhibit enhanced therapeuticefficacy relative to therapeutic antibodies known in the art, in part,due to the ability of diabody to immunospecifically bind a target cellwhich expresses a particular antigen (e.g., FcγR) at reduced levels, forexample, by virtue of the ability of the diabody to remain on the targetcell longer due to an improved avidity of the diabody-epitopeinteraction.

The diabodies of the invention with enhanced affinity and avidity forantigens (e.g., FcγRs) are particularly useful for the treatment,prevention or management of a cancer, or another disease or disorder, ina subject, wherein the FcγRs are expressed at low levels in the targetcell populations. As used herein, FcγR expression in cells is defined interms of the density of such molecules per cell as measured using commonmethods known to those skilled in the art. The molecules of theinvention comprising multiple epitope binding sites and, optionally, andFcγR (or portion thereof) preferably also have a conferred or anenhanced avidity and affinity and/or effector function in cells whichexpress a target antigen, e.g., a cancer antigen, at a density of 30,000to 20,000 molecules/cell, at a density of 20,000 to 10,000molecules/cell, at a density of 10,000 molecules/cell or less, at adensity of 5000 molecules/cell or less, or at a density of 1000molecules/cell or less. The molecules of the invention have particularutility in treatment, prevention or management of a disease or disorder,such as cancer, in a sub-population, wherein the target antigen isexpressed at low levels in the target cell population.

The molecules of the invention may also be advantageously utilized incombination with other therapeutic agents known in the art for thetreatment or prevention of diseases, such as cancer, autoimmune disease,inflammatory disorders, and infectious diseases. In a specificembodiment, molecules of the invention may be used in combination withmonoclonal or chimeric antibodies, lymphokines, or hematopoietic growthfactors (such as, e.g., IL-2, IL-3 and IL-7), which, for example, serveto increase the number or activity of effector cells which interact withthe molecules and, increase immune response. The molecules of theinvention may also be advantageously utilized in combination with one ormore drugs used to treat a disease, disorder, or infection such as, forexample anti-cancer agents, anti-inflammatory agents or anti-viralagents, e.g., as detailed in Section 5.7.

5.6.1 Cancers

The invention encompasses methods and compositions for treatment orprevention of cancer in a subject comprising administering to thesubject a therapeutically effective amount of one or more moleculescomprising multiple epitope binding domains. In some embodiments, theinvention encompasses methods and compositions for the treatment orprevention of cancer in a subject with FcγR polymorphisms such as thosehomozygous for the FγRIIIA-158V or FcγRIIIA-158F alleles. In someembodiments, the invention encompasses engineering at least one epitopebinding domain of the diabody molecule to immunospecifically bindFcγRIIIA (158F). In other embodiments, the invention encompassesengineering at least one epitope binding domain of the diabody moleculeto immunospecifically bind FcγRIIIA (158V).

The efficacy of standard monoclonal antibody therapy depends on the FcγRpolymorphism of the subject (Carton et al., 2002 Blood, 99: 754-8; Wenget al., 2003 J Clin Oncol.21(21):3940-7 both of which are incorporatedherein by reference in their entireties). These receptors are expressedon the surface of the effector cells and mediate ADCC. High affinityalleles, of the low affinity activating receptors, improve the effectorcells' ability to mediate ADCC. In contrast to relying on Fc-FcγRinteractions to effect effector function, the methods of the inventionencompass engineering molecules to immunospecifically recognize the lowaffinity activating receptors, allowing the molecules to be designed fora specific polymorphism. Alternately or additionally, the molecule ofthe invention may be engineered to comprise a variant Fc domain thatexhibits enhanced affinity to FcγR (relative to a wild type Fc domain)on effector cells. The engineered molecules of the invention providebetter immunotherapy reagents for patients regardless of their FcγRpolymorphism.

Diabody molecules engineered in accordance with the invention are testedby ADCC using either a cultured cell line or patient derived PMBC cellsto determine the ability of the Fc mutations to enhance ADCC. StandardADCC is performed using methods disclosed herein. Lymphocytes areharvested from peripheral blood using a Ficoll-Paque gradient(Pharmacia). Target cells, i.e., cultured cell lines or patient derivedcells, are loaded with Europium (PerkinElmer) and incubated witheffectors for 4 hrs at 37° C. Released Europium is detected using afluorescent plate reader (Wallac). The resulting ADCC data indicates theefficacy of the molecules of the invention to trigger NK cell mediatedcytotoxicity and establish which molecules can be tested with bothpatient samples and elutriated monocytes. Diabody molecules showing thegreatest potential for eliciting ADCC activity are then tested in anADCC assay using PBMCs from patients. PBMC from healthy donors are usedas effector cells.

Accordingly, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen by engineering the diabodymolecule to immunospecifically recognize said cancer antigen such thatthe diabody molecule is itself cytotoxic (e.g., via crosslinking ofsurface receptors leading to increased apoptosis or downregulation ofproliferative signals) and/or comprises an Fc domain, according to theinvention, and/or mediates one or more effector function (e.g., ADCC,phagocytosis). The diabodies that have been engineered according to theinvention are useful for prevention or treatment of cancer, since theyhave an cytotoxic activity (e.g., enhanced tumor cell killing and/orenhanced for example, ADCC activity or CDC activity).

Cancers associated with a cancer antigen may be treated or prevented byadministration of a diabody that binds a cancer antigen and iscytotoxic, and/or has been engineered according to the methods of theinvention to exhibit effector function. For example, but not by way oflimitation, cancers associated with the following cancer antigens may betreated or prevented by the methods and compositions of the invention:KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J. Immunol.142:32-37; Bumal, 1988, Hybridoma 7(4):407-415), ovarian carcinomaantigen (CA125) (Yu et al., 1991, Cancer Res. 51(2):48-475), prostaticacid phosphate (Tailor et al., 1990, Nucl. Acids Res. 18(1):4928),prostate specific antigen (Henttu and Vihko, 1989, Biochem. Biophys.Res. Comm. 10(2):903-910; Israeli et al., 1993, Cancer Res. 53:227-230),melanoma-associated antigen p97 (Estin et al., 1989, J. Natl. CancerInstit. 81(6):445-44), melanoma antigen gp75 (Vijayasardahl et al.,1990, J. Exp. Med. 171(4):1375-1380), high molecular weight melanomaantigen (HMW-MAA) (Natali et al., 1987, Cancer 59:55-3; Mittelman etal., 1990, J. Clin. Invest. 86:2136-2144)), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am.Soc. Clin. Oncol. 13:294), polymorphic epithelial mucin antigen, humanmilk fat globule antigen, Colorectal tumor-associated antigens such as:CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52:3402-3408), CO17-1A(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758); GICA 19-9 (Herlynet al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83:1329-1336),human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445),CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specificantigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151,3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J.Clin. Oncol. 12:1036-1044), ganglioside GM3 (Hoon et al., 1993, CancerRes. 53:5244-5250), tumor-specific transplantation type of cell-surfaceantigen (TSTA) such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and envelope antigens of RNA tumor viruses,oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46:3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245:301-304), differentiation antigen (Feizi,1985, Nature 314:53-57) such as I antigen found in fetal erthrocytes andprimary endoderm, I(Ma) found in gastric adenocarcinomas, M18 and M39found in breast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9,Myl, VIM-D5, and D₁56-22 found in colorectal cancer, TRA-1-85 (bloodgroup H), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Le^(y) found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E₁ series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Le^(a)) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Le^(b)), G49, EGF receptor, (bloodgroup ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 found incolon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2),M1:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4 foundin 4-8-cell stage embryos. In another embodiment, the antigen is a Tcell receptor derived peptide from a cutaneous T cell lymphoma (seeEdelson, 1998, The Cancer Journal 4:62).

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: Leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma;brain tumors including but not limited to, glioma, astrocytoma, brainstem glioma, ependymoma, oligodendroglioma, nonglial tumor, acousticneurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including, but notlimited to, adenocarcinoma, lobular (small cell) carcinoma, intraductalcarcinoma, medullary breast cancer, mucinous breast cancer, tubularbreast cancer, papillary breast cancer, Paget's disease, andinflammatory breast cancer; adrenal cancer, including but not limitedto, pheochromocytom and adrenocortical carcinoma; thyroid cancer such asbut not limited to papillary or follicular thyroid cancer, medullarythyroid cancer and anaplastic thyroid cancer; pancreatic cancer,including but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor;pituitary cancers including but not limited to, Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers including but not limited to, ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers, including but not limited to, squamouscell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, includingbut not limited to, squamous cell carcinoma, melanoma, adenocarcinoma,basal cell carcinoma, sarcoma, and Paget's disease; cervical cancersincluding but not limited to, squamous cell carcinoma, andadenocarcinoma; uterine cancers including but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including butnot limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including but not limited tohepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding but not limited to, adenocarcinoma; cholangiocarcinomasincluding but not limited to, pappillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including but not limitedto, squamous cell cancer, and verrucous; skin cancers including but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including but notlimited to, renal cell cancer, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer);Wilms' tumor; bladder cancers including but not limited to, transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J. B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroidand skin; including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include but not belimited to follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

In a specific embodiment, a molecule of the invention (e.g., a diabodycomprising multiple epitope binding domains and, optionally, and Fcdomain (or portion thereof)) inhibits or reduces the growth of cancercells by at least 99%, at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, at least 50%, atleast 45%, at least 40%, at least 45%, at least 35%, at least 30%, atleast 25%, at least 20%, or at least 10% relative to the growth ofcancer cells in the absence of said molecule of the invention.

In a specific embodiment, a molecule of the invention (e.g., a diabodycomprising multiple epitope binding domains and, optionally, and Fcdomain (or portion thereof)) kills cells or inhibits or reduces thegrowth of cancer cells at least 5%, at least 10%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 100% better than the parentmolecule.

5.6.2 Autoimmune Disease and Inflammatory Diseases

In some embodiments, molecules of the invention comprise an epitopebinding domain specific for FcγRIIB and or/ a variant Fc domain (orportion thereof), engineered according to methods of the invention,which Fc domain exhibits greater affinity for FcγRIIB and decreasedaffinity for FcγRIIIA and/or FcγRIIA relative to a wild-type Fc domain.Molecules of the invention with such binding characteristics are usefulin regulating the immune response, e.g., in inhibiting the immuneresponse in connection with autoimmune diseases or inflammatorydiseases. Although not intending to be bound by any mechanism of action,molecules of the invention with an affinity for FcγRIIB and/orcomprising an Fc domain with increased affinity for FcγRIIB and adecreased affinity for FcγRIIIA and/or FcγRIIA may lead to dampening ofthe activating response to FcγR and inhibition of cellularresponsiveness, and thus have therapeutic efficacy for treating and/orpreventing an autoimmune disorder.

The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an inflammatory disorderin a subject further comprising, administering to said subject atherapeutically or prophylactically effective amount of one or moreanti-inflammatory agents. The invention also provides methods forpreventing, treating, or managing one or more symptoms associated withan autoimmune disease further comprising, administering to said subjecta therapeutically or prophylactically effective amount of one or moreimmunomodulatory agents. Section 5.7 provides non-limiting examples ofanti-inflammatory agents and immunomodulatory agents.

Examples of autoimmune disorders that may be treated by administeringthe molecules of the present invention include, but are not limited to,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Ménière's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/ giant cell arteritis, ulcerativecolitis, uveitis, vasculitides such as dermatitis herpetiformisvasculitis, vitiligo, and Wegener's granulomatosis. Examples ofinflammatory disorders include, but are not limited to, asthma,encephilitis, inflammatory bowel disease, chronic obstructive pulmonarydisease (COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein inSection 2.2.2, some autoimmune disorders are associated with aninflammatory condition. Thus, there is overlap between what isconsidered an autoimmune disorder and an inflammatory disorder.Therefore, some autoimmune disorders may also be characterized asinflammatory disorders. Examples of inflammatory disorders which can beprevented, treated or managed in accordance with the methods of theinvention include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentitated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections.

Molecules of the invention comprising at least one epitope bindingdomain specific for FcγRIIB and/or a variant Fc domain with an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA can also beused to reduce the inflammation experienced by animals, particularlymammals, with inflammatory disorders. In a specific embodiment, amolecule of the invention reduces the inflammation in an animal by atleast 99%, at least 95%, at least 90%, at least 85%, at least 80%, atleast 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the inflammation in an animal,which is not administered the said molecule.

Molecules of the invention comprising at least one epitope bindingdomain specific for FcγRIIB and/or a variant Fc domain with an enhancedaffinity for FcγRIIB and a decreased affinity for FcγRIIIA can also beused to prevent the rejection of transplants.

5.6.3 Infectious Disease

The invention also encompasses methods for treating or preventing aninfectious disease in a subject comprising administering atherapeutically or prophylatically effective amount of one or moremolecules of the invention comprising at least one epitope bindingdomain specific for an infectious agent associated with said infectiousdisease. In certain embodiments, the molecules of the invention aretoxic to the infectious agent, enhance immune response against saidagent or enhance effector function against said agent, relative to theimmune response in the absence of said molecule. Infectious diseasesthat can be treated or prevented by the molecules of the invention arecaused by infectious agents including but not limited to viruses,bacteria, fungi, protozae, and viruses.

Viral diseases that can be treated or prevented using the molecules ofthe invention in conjunction with the methods of the present inventioninclude, but are not limited to, those caused by hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-I), herpes simplex type II (HSV-II),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papova virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus, small pox, Epstein Barr virus, humanimmunodeficiency virus type I (HIV-I), human immunodeficiency virus typeII (HIV-II), and agents of viral diseases such as viral miningitis,encephalitis, dengue or small pox.

Bacterial diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by bacteria include, but are not limited to,mycobacteria rickettsia, mycoplasma, neisseria, S. pneumonia, Borreliaburgdorferi (Lyme disease), Bacillus antracis (anthrax), tetanus,streptococcus, staphylococcus, mycobacterium, tetanus, pertissus,cholera, plague, diptheria, chlamydia, S. aureus and legionella.

Protozoal diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by protozoa include, but are not limited to,leishmania, kokzidioa, trypanosoma or malaria.

Parasitic diseases that can be treated or prevented using the moleculesof the invention in conjunction with the methods of the presentinvention, that are caused by parasites include, but are not limited to,chlamydia and rickettsia.

According to one aspect of the invention, molecules of the inventioncomprising at least one epitope binding domain specific for aninfectious agent exhibit an antibody effector function towards saidagent, e.g., a pathogenic protein. Examples of infectious agents includebut are not limited to bacteria (e.g., Escherichia coli, Klebsiellapneumoniae, Staphylococcus aureus, Enterococcus faecials, Candidaalbicans, Proteus vulgaris, Staphylococcus viridans, and Pseudomonasaeruginosa), a pathogen (e.g., B-lymphotropic papovavirus (LPV);Bordatella pertussis; Boma Disease virus (BDV); Bovine coronavirus;Choriomeningitis virus; Dengue virus; a virus, E. coli; Ebola; Echovirus1; Echovirus-11 (EV); Endotoxin (LPS); Enteric bacteria; Enteric Orphanvirus; Enteroviruses; Feline leukemia virus; Foot and mouth diseasevirus; Gibbon ape leukemia virus (GALV); Gram-negative bacteria;Heliobacter pylori; Hepatitis B virus (HBV); Herpes Simplex Virus;HIV-1; Human cytomegalovirus; Human coronovirus; Influenza A, B & C;Legionella; Leishmania mexicana; Listeria monocytogenes; Measles virus;Meningococcus; Morbilliviruses; Mouse hepatitis virus; Murine leukemiavirus; Murine gamma herpes virus; Murine retrovirus; Murine coronavirusmouse hepatitis virus; Mycobacterium avium-M; Neisseria gonorrhoeae;Newcastle disease virus; Parvovirus B19; Plasmodium falciparum; PoxVirus; Pseudomonas; Rotavirus; Samonella typhiurium; Shigella;Streptococci; T-cell lymphotropic virus 1; Vaccinia virus).

5.6.4 Detoxification

The invention also encompasses methods of detoxification in a subjectexposed to a toxin (e.g., a toxic drug molecule) comprisingadministering a therapeutically or prophylatically effective amount ofone or more molecules of the invention comprising at least one epitopebinding domain specific for the toxic drug molecule. In certainembodiments, binding of a molecule of the invention to the toxin reducesor eliminates the adverse physiological effect of said toxin. In yetother embodiments, binding of a diabody of the invention to the toxinincreases or enhances elimination, degradation or neutralization of thetoxin relative to elimination, degradation or neutralization in theabsence of said diabody. Immunotoxicotherapy in accordance with themethods of the invention can be used to treat overdoses or exposure todrugs including, but not limited to, digixin, PCP, cocaine, colchicine,and tricyclic antidepressants.

5.7 Combination Therapy

The invention further encompasses administering the molecules of theinvention in combination with other therapies known to those skilled inthe art for the treatment or prevention of cancer, autoimmune disease,infectious disease or intoxication, including but not limited to,current standard and experimental chemotherapies, hormonal therapies,biological therapies, immunotherapies, radiation therapies, or surgery.In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or more agents, therapeutic antibodies or other agentsknown to those skilled in the art for the treatment and/or prevention ofcancer, autoimmune disease, infectious disease or intoxication.

In certain embodiments, one or more molecule of the invention isadministered to a mammal, preferably a human, concurrently with one ormore other therapeutic agents useful for the treatment of cancer. Theterm “concurrently” is not limited to the administration of prophylacticor therapeutic agents at exactly the same time, but rather it is meantthat a molecule of the invention and the other agent are administered toa mammal in a sequence and within a time interval such that the moleculeof the invention can act together with the other agent to provide anincreased benefit than if they were administered otherwise. For example,each prophylactic or therapeutic agent (e.g., chemotherapy, radiationtherapy, hormonal therapy or biological therapy) may be administered atthe same time or sequentially in any order at different points in time;however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute. In various embodiments, the prophylactic or therapeutic agentsare administered less than 1 hour apart, at about 1 hour apart, at about1 hour to about 2 hours apart, at about 2 hours to about 3 hours apart,at about 3 hours to about 4 hours apart, at about 4 hours to about 5hours apart, at about 5 hours to about 6 hours apart, at about 6 hoursto about 7 hours apart, at about 7 hours to about 8 hours apart, atabout 8 hours to about 9 hours apart, at about 9 hours to about 10 hoursapart, at about 10 hours to about 11 hours apart, at about 11 hours toabout 12 hours apart, no more than 24 hours apart or no more than 48hours apart. In preferred embodiments, two or more components areadministered within the same patient visit.

In other embodiments, the prophylactic or therapeutic agents areadministered at about 2 to 4 days apart, at about 4 to 6 days apart, atabout 1 week part, at about 1 to 2 weeks apart, or more than 2 weeksapart. In preferred embodiments, the prophylactic or therapeutic agentsare administered in a time frame where both agents are still active. Oneskilled in the art would be able to determine such a time frame bydetermining the half life of the administered agents.

In certain embodiments, the prophylactic or therapeutic agents of theinvention are cyclically administered to a subject. Cycling therapyinvolves the administration of a first agent for a period of time,followed by the administration of a second agent and/or third agent fora period of time and repeating this sequential administration. Cyclingtherapy can reduce the development of resistance to one or more of thetherapies, avoid or reduce the side effects of one of the therapies,and/or improves the efficacy of the treatment.

In certain embodiments, prophylactic or therapeutic agents areadministered in a cycle of less than about 3 weeks, about once every twoweeks, about once every 10 days or about once every week. One cycle cancomprise the administration of a therapeutic or prophylactic agent byinfusion over about 90 minutes every cycle, about 1 hour every cycle,about 45 minutes every cycle. Each cycle can comprise at least 1 week ofrest, at least 2 weeks of rest, at least 3 weeks of rest. The number ofcycles administered is from about 1 to about 12 cycles, more typicallyfrom about 2 to about 10 cycles, and more typically from about 2 toabout 8 cycles.

In yet other embodiments, the therapeutic and prophylactic agents of theinvention are administered in metronomic dosing regimens, either bycontinuous infusion or frequent administration without extended restperiods. Such metronomic administration can involve dosing at constantintervals without rest periods. Typically the therapeutic agents, inparticular cytotoxic agents, are used at lower doses. Such dosingregimens encompass the chronic daily administration of relatively lowdoses for extended periods of time. In preferred embodiments, the use oflower doses can minimize toxic side effects and eliminate rest periods.In certain embodiments, the therapeutic and prophylactic agents aredelivered by chronic low-dose or continuous infusion ranging from about24 hours to about 2 days, to about 1 week, to about 2 weeks, to about 3weeks to about 1 month to about 2 months, to about 3 months, to about 4months, to about 5 months, to about 6 months. The scheduling of suchdose regimens can be optimized by the skilled oncologist.

In other embodiments, courses of treatment are administered concurrentlyto a mammal, i.e., individual doses of the therapeutics are administeredseparately yet within a time interval such that molecules of theinvention can work together with the other agent or agents. For example,one component may be administered one time per week in combination withthe other components that may be administered one time every two weeksor one time every three weeks. In other words, the dosing regimens forthe therapeutics are carried out concurrently even if the therapeuticsare not administered simultaneously or within the same patient visit.

When used in combination with other prophylactic and/or therapeuticagents, the molecules of the invention and the prophylactic and/ortherapeutic agent can act additively or, more preferably,synergistically. In one embodiment, a molecule of the invention isadministered concurrently with one or more therapeutic agents in thesame pharmaceutical composition. In another embodiment, a molecule ofthe invention is administered concurrently with one or more othertherapeutic agents in separate pharmaceutical compositions. In stillanother embodiment, a molecule of the invention is administered prior toor subsequent to administration of another prophylactic or therapeuticagent. The invention contemplates administration of a molecule of theinvention in combination with other prophylactic or therapeutic agentsby the same or different routes of administration, e.g., oral andparenteral. In certain embodiments, when a molecule of the invention isadministered concurrently with another prophylactic or therapeutic agentthat potentially produces adverse side effects including, but notlimited to, toxicity, the prophylactic or therapeutic agent canadvantageously be administered at a dose that falls below the thresholdthat the adverse side effect is elicited.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference (56^(th) ed., 2002).

5.7.1 Anti-Cancer Agents

In a specific embodiment, the methods of the invention encompass theadministration of one or more molecules of the invention with one ormore therapeutic agents used for the treatment and/or prevention ofcancer. In one embodiment, angiogenesis inhibitors may be administeredin combination with the molecules of the invention. Angiogenesisinhibitors that can be used in the methods and compositions of theinvention include but are not limited to: Angiostatin (plasminogenfragment); antiangiogenic antithrombin III; Angiozyme; ABT-627; Bay12-9566; Benefin; Bevacizumab; BMS-275291; cartilage-derived inhibitor(CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; CombretastatinA-4; Endostatin (collagen XVIII fragment); Fibronectin fragment;Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment;HMV833; Human chorionic gonadotropin (hCG); IM-862; Interferonalpha/beta/gamma; Interferon inducible protein (IP-10); Interleukin-12;Kringle 5 (plasminogen fragment); Marimastat; Metalloproteinaseinhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS 27023A); MoAbIMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placental ribonucleaseinhibitor; Plasminogen activator inhibitor; Platelet factor-4 (PF4);Prinomastat; Prolactin 16 kDa fragment; Proliferin-related protein(PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS 3304; SU5416; SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate;thalidomide; Thrombospondin-1 (TSP-1); TNP-470; Transforming growthfactor-beta (TGF-b); Vasculostatin; Vasostatin (calreticulin fragment);ZD6126; ZD 6474; farnesyl transferase inhibitors (FTI); andbisphosphonates.

Anti-cancer agents that can be used in combination with the molecules ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflomithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-I a; interferon gamma-I b;iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole;leuprolide acetate; liarozole hydrochloride; lometrexol sodium;lomustine; losoxantrone hydrochloride; masoprocol; maytansine;mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium;metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride;mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspennine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to ZENAPAX® (daclizumab) (RochePharmaceuticals, Switzerland) which is an immunosuppressive, humanizedanti-CD25 monoclonal antibody for the prevention of acute renalallograft rejection; PANOREX· which is a murine anti-17-IA cell surfaceantigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murineanti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™which is a humanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); LYMPHOCIDE™ which is a humanizedanti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatied anti-CD80 antibody (IDECPharmi/Mitsubishi); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm); D2E7is a humanized anti-TNF-α antibody (CAT/BASF); CDP870 is a humanizedanti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanizedanti-TNF-a IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgGantibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 is a human anti-TGF-β₂ antibody (Cambridge Ab Tech).Other examples of therapeutic antibodies that can be used in accordancewith the invention are presented in Table 8. TABLE 8 Anti-cancertherapeutic antibodies Company Product Disease Target Abgenix ABX-EGFCancer EGF receptor AltaRex OvaRex ovarian cancer tumor antigen CA125BravaRex metastatic tumor antigen MUC1 cancers Antisoma Theragyn ovariancancer PEM antigen (pemtumomabytrrium- 90) Therex breast cancer PEMantigen Boehringer Blvatuzumab head & neck CD44 Ingelheim cancerCentocor/J&J Panorex Colorectal 17-1A cancer ReoPro PTCA gp IIIb/IIIaReoPro Acute MI gp IIIb/IIIa ReoPro Ischemic stroke gp IIIb/IIIa CorixaBexocar NHL CD20 CRC MAb, idiotypic 105AD7 colorectal cancer gp72Technology vaccine Crucell Anti-EpCAM cancer Ep-CAM Cytoclonal MAb, lungcancer non-small cell NA lung cancer Genentech Herceptin metastaticbreast HER-2 cancer Herceptin early stage HER-2 breast cancer RituxanRelapsed/refractory CD20 low-grade or follicular NHL Rituxanintermediate & CD20 high-grade NHL MAb-VEGF NSCLC, VEGF metastaticMAb-VEGF Colorectal VEGF cancer, metastatic AMD Fab age-related CD18macular degeneration E-26 (2^(nd) gen. IgE) allergic asthma IgE &rhinitis IDEC Zevalin (Rituxan + yttrium- low grade of CD20 90)follicular, relapsed or refractory, CD20-positive, B-cell NHL andRituximab- refractory NHL ImClone Cetuximab + innotecan refractory EGFreceptor colorectal carcinoma Cetuximab + cisplatin & newly diagnosedEGF receptor or recurrent head radiation & neck cancer Cetuximab +gemcitabine newly diagnosed EGF receptor metastatic pancreatic carcinomaCetuximab + cisplatin + 5FU recurrent or EGF receptor or Taxolmetastatic head & neck cancer Cetuximab + carboplatin + paclitaxel newlydiagnosed EGF receptor non-small cell lung carcinoma Cetuximab +cisplatin head & neck EGF receptor cancer (extensive incurable local-regional disease & distant metasteses) Cetuximab + radiation locallyadvanced EGF receptor head & neck carcinoma BEC2 + Bacillus small celllung mimics ganglioside Calmette Guerin carcinoma GD3 BEC2 + Bacillusmelanoma mimics ganglioside Calmette Guerin GD3 IMC-1C11 colorectalcancer VEGF-receptor with liver metasteses ImmonoGen nuC242-DM1Colorectal, nuC242 gastric, and pancreatic cancer ImmunoMedicsLymphoCide Non-Hodgkins CD22 lymphoma LymphoCide Y-90 Non-Hodgkins CD22lymphoma CEA-Cide metastatic solid CEA tumors CEA-Cide Y-90 metastaticsolid CEA tumors CEA-Scan (Tc-99m- colorectal cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan (Tc-99m- Breast cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan (Tc-99m- lung cancer CEA labeledarcitumomab) (radioimaging) CEA-Scan (Tc-99m- intraoperative CEA labeledarcitumomab) tumors (radio imaging) LeukoScan (Tc-99m- soft tissue CEAlabeled sulesomab) infection (radioimaging) LymphoScan (Tc-99m-lymphomas CD22 labeled) (radioimaging) AFP-Scan (Tc-99m- liver 7gem-cell AFP labeled) cancers (radioimaging) Intracel HumaRAD-HN (+yttrium- head & neck NA 90) cancer HumaSPECT colorectal NA imagingMedarex MDX-101 (CTLA-4) Prostate and CTLA-4 other cancers MDX-210(her-2 Prostate cancer HER-2 overexpression) MDX-210/MAK Cancer HER-2MedImmune Vitaxin Cancer αvβ₃ Merck KGaA MAb 425 Various cancers EGFreceptor IS-IL-2 Various cancers Ep-CAM Millennium Campath chronic CD52(alemtuzumab) lymphocytic leukemia NeoRx CD20-streptavidin (+ biotin-Non-Hodgkins CD20 yttrium 90) lymphoma Avidicin (albumin + NRLU13)metastatic NA cancer Peregrine Oncolym (+ iodine-131) Non-HodgkinsHLA-DR 10 beta lymphoma Cotara (+ iodine-131) unresectableDNA-associated malignant proteins glioma Pharmacia C215 (+staphylococcal pancreatic NA Corporation enterotoxin) cancer MAb,lung/kidney lung & kidney NA cancer cancer nacolomab tafenatox colon &NA (C242 + staphylococcal pancreatic enterotoxin) cancer Protein DesignNuvion T cell CD3 Labs malignancies SMART M195 AML CD33 SMART 1D10 NHLHLA-DR antigen Titan CEAVac colorectal CEA cancer, advanced TriGemmetastatic GD2-ganglioside melanoma & small cell lung cancer TriAbmetastatic breast MUC-1 cancer Trilex CEAVac colorectal CEA cancer,advanced TriGem metastatic GD2-ganglioside melanoma & small cell lungcancer TriAb metastatic breast MUC-1 cancer Viventia NovoMAb-G2Non-Hodgkins NA Biotech radiolabeled lymphoma Monopharm C colorectal &SK-1 antigen pancreatic carcinoma GlioMAb-H (+ gelonin gliorna, NAtoxin) melanoma & neuroblastoma Xoma Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLING-1 adenomcarcinoma Ep-CAM5.7.2 Immunomodulatory Agents and Anti-Inflammatory Agents

The present invention provides methods of treatment for autoimmunediseases and inflammatory diseases comprising administration of themolecules of the invention in conjunction with other treatment agents.Examples of immunomodulatory agents include, but are not limited to,methothrexate, ENBREL, REMICADE™, leflunomide, cyclophosphamide,cyclosporine A, and macrolide antibiotics (e.g., FK506 (tacrolimus)),methylprednisolone (MP), corticosteroids, steriods, mycophenolatemofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,and cytokine receptor modulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxgenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

A non-limiting example of the antibodies that can be used for thetreatment or prevention of inflammatory disorders in conjunction withthe molecules of the invention is presented in Table 9, and anon-limiting example of the antibodies that can used for the treatmentor prevention of autoimmune disorder is presented in Table 10. TABLE 9Therapeutic antibodies for the treatment of inflammatory diseasesAntibody Target Product Name Antigen Type Isotype Sponsors Indication5G1.1 Complement Humanized IgG Alexion Rheumatoid (C5) Pharm IncArthritis 5G1.1 Complement Humanized IgG Alexion SLE (C5) Pharm Inc5G1.1 Complement Humanized IgG Alexion Nephritis (C5) Pharm Inc 5G1.1-SCComplement Humanized ScFv Alexion Cardiopulmonary (C5) Pharm Inc Bypass5G1.1-SC Complement Humanized ScFv Alexion Myocardial (C5) Pharm IncInfarction 5G1.1-SC Complement Humanized ScFv Alexion Angioplasty (C5)Pharm Inc ABX-CBL CBL Human Abgenix Inc GvHD ABX-CBL CD147 Murine IgGAbgenix Inc Allograft rejection ABX-IL8 IL-8 Human IgG2 Abgenix IncPsoriasis Antegren VLA-4 Humanized IgG Athena/Elan Multiple SclerosisAnti- CD11a Humanized IgG1 Genentech Psoriasis CD11a Inc/Xoma Anti- CD18Humanized Fab'2 Genentech Inc Myocardial CD18 infarction Anti- CD18Murine Fab'2 Pasteur- Allograft rejection LFA1 Merieux/ ImmunotechAntova CD40L Humanized IgG Biogen Allograft rejection Antova CD40LHumanized IgG Biogen SLE BTI-322 CD2 Rat IgG Medimmune GvHD, PsoriasisInc CDP571 TNF-alpha Humanized IgG4 Celltech Crohn's CDP571 TNF-alphaHumanized IgG4 Celltech Rheumatoid Arthritis CDP850 E-selectin HumanizedCelltech Psoriasis Corsevin M Fact VII Chimeric Centocor AnticoagulantD2E7 TNF-alpha Human CAT/BASF Rheumatoid Arthritis Hu23F2G CD11/18Humanized ICOS Pharm Multiple Sclerosis Inc Hu23F2G CD11/18 HumanizedIgG ICOS Pharm Stroke Inc IC14 CD14 ICOS Pharm Toxic shock Inc ICM3ICAM-3 Humanized ICOS Pharm Psoriasis Inc IDEC-114 CD80 Primatised IDECPsoriasis Pharm/Mitsubishi IDEC-131 CD40L Humanized IDEC SLE Pharm/EisaiIDEC-131 CD40L Humanized IDEC Multiple Sclerosis Pharm/Eisai IDEC-151CD4 Primatised IgG1 IDEC Rheumatoid Pharm/Glaxo Arthritis SmithKlineIDEC-152 CD23 Primatised IDEC Pharm Asthma/Allergy Infliximab TNF-alphaChimeric IgG1 Centocor Rheumatoid Arthritis Infliximab TNF-alphaChimeric IgG1 Centocor Crohn's LDP-01 beta2- Humanized IgG MillenniumStroke integrin Inc (LeukoSite Inc.) LDP-01 beta2- Humanized IgGMillennium Allograft rejection integrin Inc (LeukoSite Inc.) LDP-02alpha4beta7 Humanized Millennium Ulcerative Colitis Inc (LeukoSite Inc.)MAK- TNF alpha Murine Fab'2 Knoll Pharm, Toxic shock 195F BASF MDX-33CD64 (FcR) Human Medarex/Centeon Autoimmune haematogical disorders MDX-CD4 Human IgG Medarex/Eisai/ Rheumatoid CD4 Genmab Arthritis MEDI-507CD2 Humanized Medimmune Psoriasis Inc MEDI-507 CD2 Humanized MedimmuneGvHD Inc OKT4A CD4 Humanized IgG Ortho Biotech Allograft rejectionOrthoClone CD4 Humanized IgG Ortho Biotech Autoimmune OKT4A diseaseOrthoclone/ CD3 Murine mIgG2a Ortho Biotech Allograft rejection anti-CD3OKT3 RepPro/ gpIIbIIIa Chimeric Fab Centocor/Lilly Complications ofAbciximab coronary angioplasty rhuMab- IgE Humanized IgG1Genentech/Novartis/ Asthma/Allergy E25 Tanox Biosystems SB-240563 IL5Humanized GlaxoSmithKline Asthma/Allergy SB-240683 IL-4 HumanizedGlaxoSmithKline Asthma/Allergy SCH55700 IL-5 Humanized Celltech/ScheringAsthma/Allergy Simulect CD25 Chimeric IgG1 Novartis Allograft rejectionPharm SMART CD3 Humanized Protein Autoimmune a-CD3 Design Lab diseaseSMART CD3 Humanized Protein Allograft rejection a-CD3 Design Lab SMARTCD3 Humanized IgG Protein Psoriasis a-CD3 Design Lab Zenapax CD25Humanized IgG1 Protein Allograft rejection Design Lab/Hoffman- La Roche

TABLE 10 Therapeutic antibodies for the treatment of autoimmunedisorders Antibody Indication Target Antigen ABX-RB2 antibody to CBLantigen on T cells, B cells and NK cells fully human antibody from theXenomouse 5c8 (Anti CD-40 an Phase II trials were halted in October,CD-40 antigen antibody) 1999 examine “adverse events” IDEC 131 systemiclupus erythyematous anti CD40 (SLE) humanized IDEC 151 rheumatoidarthritis primatized; anti-CD4 IDEC 152 Asthma primatized; anti-CD23IDEC 114 Psoriasis primatized anti-CD80 MEDI-507 rheumatoid arthritis;multiple anti-CD2 sclerosis Crohn's disease Psoriasis LDP-02 (anti-b7inflammatory bowel disease a4b7 integrin receptor on white mAb) Chron'sdisease blood cells (leukocytes) ulcerative colitis SMART Anti-autoimmune disorders Anti-Gamma Interferon Gamma Interferon antibodyVerteportin rheumatoid arthritis MDX-33 blood disorders caused bymonoclonal antibody against FcRI autoimmune reactions receptorsIdiopathic Thrombocytopenia Purpurea (ITP) autoimmune hemolytic anemiaMDX-CD4 treat rheumatoid arthritis and other monoclonal antibody againstCD4 autoimmunity receptor molecule VX-497 autoimmune disorders inhibitorof inosine monophosphate multiple sclerosis dehydrogenase rheumatoidarthritis (enzyme needed to make new RNA inflammatory bowel disease andDNA lupus used in production of nucleotides psoriasis needed forlymphocyte proliferation) VX-740 rheumatoid arthritis inhibitor of ICEinterleukin-1 beta (converting enzyme controls pathways leading toaggressive immune response) VX-745 specific to inflammation inhibitor ofP38MAP kinase involved in chemical signalling of mitogen activatedprotein kinase immune response onset and progression of inflammationEnbrel (etanercept) targets TNF (tumor necrosis factor) IL-8 fully humanmonoclonal antibody against IL-8 (interleukin 8) Apogen MP4 recombinantantigen selectively destroys disease associated T-cells inducesapoptosis T-cells eliminated by programmed cell death no longer attackbody's own cells specific apogens target specific T- cells5.7.3 Agents for Use in the Treatment of Infectious Disease

In some embodiments, the molecules of the invention may be administeredin combination with a therapeutically or prophylactically effectiveamount of one or additional therapeutic agents known to those skilled inthe art for the treatment and/or prevention of an infectious disease.The invention contemplates the use of the molecules of the invention incombination with antibiotics known to those skilled in the art for thetreatment and or prevention of an infectious disease. Antibiotics thatcan be used in combination with the molecules of the invention include,but are not limited to, macrolide (e.g., tobramycin (Tobi®)), acephalosporin (e.g., cephalexin (Keflex®), cephradine (Velosef®),cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime(Suprax®) or cefadroxil (Duricef®)), a clarithromycin (e.g.,clarithromycin (Biaxin®)), an erythromycin (e.g., erythromycin(EMycin®)), a penicillin (e.g., penicillin V (V-Cillin K® or Pen VeeK®)) or a quinolone (e.g., ofloxacin (Floxin®), ciprofloxacin (Cipro®)or norfloxacin (Noroxin®)), aminoglycoside antibiotics (e.g., apramycin,arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin,undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, andspectinomycin), amphenicol antibiotics (e.g., azidamfenicol,chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics(e.g., rifamide and rifampin), carbacephems (e.g., loracarbef),carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g.,cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefozopran,cefpimizole, cefpiramide, and cefpirome), cephamycins (e.g.,cefbuperazone, cefinetazole, and cefminox), monobactams (e.g.,aztreonam, carumonam, and tigemonam), oxacephems (e.g., flomoxef, andmoxalactam), penicillins (e.g., amdinocillin, amdinocillin pivoxil,amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillinsodium, epicillin, fenbenicillin, floxacillin, penamccillin, penethamatehydriodide, penicillin o-benethamine, penicillin 0, penicillin V,penicillin V benzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin, clinafloxacin,flumequine, and grepagloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

In certain embodiments, the molecules of the invention can beadministered in combination with a therapeutically or prophylacticallyeffective amount of one or more antifingal agents. Antifungal agentsthat can be used in combination with the molecules of the inventioninclude but are not limited to amphotericin B, itraconazole,ketoconazole, fluconazole, intrathecal, flucytosine, miconazole,butoconazole, clotrimazole, nystatin, terconazole, tioconazole,ciclopirox, econazole, haloprogrin, naftifine, terbinafine,undecylenate, and griseofuldin.

In some embodiments, the molecules of the invention can be administeredin combination with a therapeutically or prophylactically effectiveamount of one or more anti-viral agent. Useful anti-viral agents thatcan be used in combination with the molecules of the invention include,but are not limited to, protease inhibitors, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors and nucleoside analogs. Examples of antiviral agents includebut are not limited to zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin, as well as foscarnet,amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir,ritonavir, the alpha-interferons; adefovir, clevadine, entecavir,pleconaril.

5.8 Vaccine Therapy

The invention further encompasses using a composition of the inventionto induce an immune response against an antigenic or immunogenic agent,including but not limited to cancer antigens and infectious diseaseantigens (examples of which are disclosed infra). The vaccinecompositions of the invention comprise one or more antigenic orimmunogenic agents to which an immune response is desired, wherein theone or more antigenic or immunogenic agents is coated with a variantantibody of the invention that has an enhanced affinity to FcγRIIIA. Thevaccine compositions of the invention are particularly effective ineliciting an immune response, preferably a protective immune responseagainst the antigenic or immunogenic agent.

In some embodiments, the antigenic or immunogenic agent in the vaccinecompositions of the invention comprise a virus against which an immuneresponse is desired. The viruses may be recombinant or chimeric, and arepreferably attenuated. Production of recombinant, chimeric, andattenuated viruses may be performed using standard methods known to oneskilled in the art. The invention encompasses a live recombinant viralvaccine or an inactivated recombinant viral vaccine to be formulated inaccordance with the invention. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In a specific embodiment, the recombinant virus is non-pathogenic to thesubject to which it is administered. In this regard, the use ofgenetically engineered viruses for vaccine purposes may require thepresence of attenuation characteristics in these strains. Theintroduction of appropriate mutations (e.g., deletions) into thetemplates used for transfection may provide the novel viruses withattenuation characteristics. For example, specific missense mutationswhich are associated with temperature sensitivity or cold adaptation canbe made into deletion mutations. These mutations should be more stablethan the point mutations associated with cold or temperature sensitivemutants and reversion frequencies should be extremely low. RecombinantDNA technologies for engineering recombinant viruses are known in theart and encompassed in the invention. For example, techniques formodifying negative strand RNA viruses are known in the art, see, e.g.,U.S. Pat. No. 5,166,057, which is incorporated herein by reference inits entirety.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed for use in the intradermal vaccine formulations of theinvention. Such viruses would go through only one or a few rounds ofreplication within the host. When used as a vaccine, the recombinantvirus would go through limited replication cycle(s) and induce asufficient level of immune response but it would not go further in thehuman host and cause disease. Alternatively, inactivated (killed) virusmay be formulated in accordance with the invention. Inactivated vaccineformulations may be prepared using conventional techniques to “kill” thechimeric viruses. Inactivated vaccines are “dead” in the sense thattheir infectivity has been destroyed. Ideally, the infectivity of thevirus is destroyed without affecting its immunogenicity. In order toprepare inactivated vaccines, the chimeric virus may be grown in cellculture or in the allantois of the chick embryo, purified by zonalultracentrifugation, inactivated by formaldehyde or β-propiolactone, andpooled.

In certain embodiments, completely foreign epitopes, including antigensderived from other viral or non-viral pathogens can be engineered intothe virus for use in the intradermal vaccine formulations of theinvention. For example, antigens of non-related viruses such as HIV(gp160, gp120, gp41) parasite antigens (e.g., malaria), bacterial orfungal antigens or tumor antigens can be engineered into the attenuatedstrain.

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in the intradermal vaccineformulations. Preferably, heterologous gene sequences are moieties andpeptides that act as biological response modifiers. Preferably, epitopesthat induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention include, but are not limited to, influenza andparainfluenza hemagglutinin neuraminidase and fusion glycoproteins suchas the HN and F genes of human PIV3. In yet another embodiment,heterologous gene sequences that can be engineered into the chimericviruses include those that encode proteins with immuno-modulatingactivities. Examples of immuno-modulating proteins include, but are notlimited to, cytokines, interferon type 1, gamma interferon, colonystimulating factors, interleukin-1, -2, -4, -5, -6, -12, and antagonistsof these agents.

In yet other embodiments, the invention encompasses pathogenic cells orviruses, preferably attenuated viruses, which express the variantantibody on their surface.

In alternative embodiments, the vaccine compositions of the inventioncomprise a fusion polypeptide wherein an antigenic or immunogenic agentis operatively linked to a variant antibody of the invention that has anenhanced affinity for FcγRIIIA. Engineering fusion polypeptides for usein the vaccine compositions of the invention is performed using routinerecombinant DNA technology methods and is within the level of ordinaryskill.

The invention further encompasses methods to induce tolerance in asubject by administering a composition of the invention. Preferably acomposition suitable for inducing tolerance in a subject, comprises anantigenic or immunogenic agent coated with a variant antibody of theinvention, wherein the variant antibody has a higher affinity toFcγRIIB. Although not intending to be bound by a particular mechanism ofaction, such compositions are effective in inducing tolerance byactivating the FcγRIIB mediatated inhibitory pathway.

5.9 Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising molecules of the invention (i.e., diabodies) comprisingmultiple epitope binding domains and, optionally, an Fc domain (orportion thereof). The invention also provides methods of treatment,prophylaxis, and amelioration of one or more symptoms associated with adisease, disorder or infection by administering to a subject aneffective amount of a fusion protein or a conjugated molecule of theinvention, or a pharmaceutical composition comprising a fusion proteinor a conjugated molecule of the invention. In a preferred aspect, anantibody, a fusion protein, or a conjugated molecule, is substantiallypurified (i.e., substantially free from substances that limit its effector produce undesired side-effects). In a specific embodiment, thesubject is an animal, preferably a mammal such as non-primate (e.g.,cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkeysuch as, a cynomolgous monkey and a human). In a preferred embodiment,the subject is a human. In yet another preferred embodiment, theantibody of the invention is from the same species as the subject.

Various delivery systems are known and can be used to administer acomposition comprising molecules of the invention, e.g., encapsulationin liposomes, microparticles, microcapsules, recombinant cells capableof expressing the antibody or fusion protein, receptor-mediatedendocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432),construction of a nucleic acid as part of a retroviral or other vector,etc. Methods of administering a molecule of the invention include, butare not limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the molecules of the invention are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example, by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety.

The invention also provides that the molecules of the invention, arepackaged in a hermetically sealed container such as an ampoule orsachette indicating the quantity of antibody. In one embodiment, themolecules of the invention are supplied as a˜dry sterilized lyophilizedpowder or water free concentrate in a hermetically sealed container andcan be reconstituted, e.g., with water or saline to the appropriateconcentration for administration to a subject. Preferably, the moleculesof the invention are supplied as a dry sterile lyophilized powder in ahermetically sealed container at a unit dosage of at least 5 mg, morepreferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilizedmolecules of the invention should be stored at between 2 and 8° C. intheir original container and the molecules should be administered within12 hours, preferably within 6 hours, within 5 hours, within 3 hours, orwithin 1 hour after being reconstituted. In an alternative embodiment,molecules of the invention are supplied in liquid form in a hermeticallysealed container indicating the quantity and concentration of themolecule, fusion protein, or conjugated molecule. Preferably, the liquidform of the molecules of the invention are supplied in a hermeticallysealed container at least 1 mg/ml, more preferably at least 2.5 mg/ml,at least 5 mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15mg/kg, at least 25 mg/ml, at least 50 mg/ml, at least 100 mg/ml, atleast 150 mg/ml, at least 200 mg/ml of the molecules.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For diabodies encompassed by the invention, the dosage administered to apatient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. The dosage andfrequency of administration of diabodies of the invention may be reducedor altered by enhancing uptake and tissue penetration of the diabodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the molecules of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the molecules of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidmolecules are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a molecule of the invention, care must be taken touse materials to which the molecule does not absorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more molecules of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al., 1996, “IntratumoralRadioimmunotheraphy of a Human Colon Cancer Xenograft Using aSustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.,1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al., 1997, “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton, 1987, CRC Crit. Ref Biomed Eng. 14:20; Buchwald et al., 1980,Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med 321:574). Inanother embodiment, polymeric materials can be used to achievecontrolled release of antibodies (see e.g., Medical Applications ofControlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.(1974); Controlled Drug Bioavailability, Drug Product Design andPerformance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger andPeppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; See alsoLevy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat.No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154;and PCT Publication No. WO 99/20253). Examples of polymers used insustained release formulations include, but are not limited to,poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yetanother embodiment, a controlled release system can be placed inproximity of the therapeutic target (e.g., the lungs), thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).In another embodiment, polymeric compositions useful as controlledrelease implants are used according to Dunn et al. (See U.S. Pat. No.5,945,155). This particular method is based upon the therapeutic effectof the in situ controlled release of the bioactive material from thepolymer system. The implantation can generally occur anywhere within thebody of the patient in need of therapeutic treatment. In anotherembodiment, a non-polymeric sustained delivery system is used, whereby anon-polymeric implant in the body of the subject is used as a drugdelivery system. Upon implantation in the body, the organic solvent ofthe implant will dissipate, disperse, or leach from the composition intosurrounding tissue fluid, and the non-polymeric material will graduallycoagulate or precipitate to form a solid, microporous matrix (See U.S.Pat. No. 5,888,533).

Controlled release systems are discussed in the review by Langer (1990,Science 249:1527-1533). Any technique known to one of skill in the artcan be used to produce sustained release formulations comprising one ormore therapeutic agents of the invention. See, e.g., U.S. Pat. No.4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698;Ning et al., 1996, Radiotherapy & Oncology 39:179-189; Song et al.,1995, PDA Journal of Pharmaceutical Science & Technology 50:372-397;Cleek et al., 1997, Pro. Int'l. Symp. Control. Rel. Bioact. Mater.24:853-854; and Lam et al., 1997, Proc. Int'l. Symp. Control Rel.Bioact. Mater. 24:759-760, each of which is incorporated herein byreference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding a diabody of the invention, the nucleic acid canbe administered in vivo to promote expression of its encoded diabody, byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of molecules of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with molecules of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the molecules used for treatment may increase or decrease overthe course of a particular treatment.

5.9.1 Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of one or more molecules of theinvention and a pharmaceutically acceptable carrier.

The invention also encompasses pharmaceutical compositions comprising adiabody molecule of the invention and a therapeutic antibody (e.g.,tumor specific monoclonal antibody) that is specific for a particularcancer antigen, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

5.9.2 Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingmolecules of the invention, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, 1993,Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding a diabody of the invention, said nucleic acids being partof an expression vector that expresses the antibody in a suitable host.In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the antibody coding region,said promoter being inducible or constitutive, and, optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the antibody coding sequences and any otherdesired sequences are flanked by regions that promote homologousrecombination at a desired site in the genome, thus providing forintrachromosomal expression of the antibody encoding nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; andZijlstra et al., 1989, Nature 342:435-438).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expresses the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a an antigen subject to receptor-mediatedendocytosis (See, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-an antigencomplexes can be formed in which the an antigen comprises a fusogenicviral peptide to disrupt endosomes, allowing the nucleic acid to avoidlysosomal degradation. In yet another embodiment, the nucleic acid canbe targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (See, e.g., PCT Publications WO 92/06180;WO 92/22635; WO92/20316; WO93/14188; WO 93/20221). Alternatively, thenucleic acid can be introduced intracellularly and incorporated withinhost cell DNA for expression, by homologous recombination (Koller andSmithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra etal., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding a molecule of the invention (e.g., a diabody or afusion protein) are used. For example, a retroviral vector can be used(See Miller et al., 1993, Meth. Enzymol. 217:581-599). These retroviralvectors contain the components necessary for the correct packaging ofthe viral genome and integration into the host cell DNA. The nucleicacid sequences encoding the antibody or a fusion protein to be used ingene therapy are cloned into one or more vectors, which facilitatesdelivery of the nucleotide sequence into a subject. More detail aboutretroviral vectors can be found in Boesen et al., (1994, Biotherapy6:291-302), which describes the use of a retroviral vector to deliverthe mdr 1 gene to hematopoietic stem cells in order to make the stemcells more resistant to chemotherapy. Other references illustrating theuse of retroviral vectors in gene therapy are: Clowes et al., 1994, J.Clin. Invest. 93:644-651; Klein et al., 1994, Blood 83:1467-1473;Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossmanand Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson (CurrentOpinion in Genetics and Development 3:499-503, 1993, present a review ofadenovirus-based gene therapy. Bout et al., (Human Gene Therapy, 5:3-10,1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g.,Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.204:289-300 and U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to, transfection, electroporation,microinjection, infection with a viral or bacteriophage vector,containing the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (See, e.g., Loeffler and Behr, 1993, Meth. Enzymol.217:599-618, Cohen et al., 1993, Meth. Enzymol. 217:618-644; and Clin.Pharma. Ther. 29:69-92, 1985) and may be used in accordance with thepresent invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (See e.g., PCT Publication WO94/08598; Stemple and Anderson, 1992, Cell 7 1:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

5.9.3 Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with the molecules of the invention.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a disease can also be included in thepharmaceutical pack or kit. The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more molecules of theinvention. In another embodiment, a kit further comprises one or moreother prophylactic or therapeutic agents useful for the treatment ofcancer, in one or more containers. In another embodiment, a kit furthercomprises one or more cytotoxic antibodies that bind one or more cancerantigens associated with cancer. In certain embodiments, the otherprophylactic or therapeutic agent is a chemotherapeutic. In otherembodiments, the prophylactic or therapeutic agent is a biological orhormonal therapeutic.

5.10 Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions, prophylactic, ortherapeutic agents of the invention are preferably tested in vitro, in acell culture system, and in an animal model organism, such as a rodentanimal model system, for the desired therapeutic activity prior to usein humans. For example, assays which can be used to determine whetheradministration of a specific pharmaceutical composition is desired,include cell culture assays in which a patient tissue sample is grown inculture, and exposed to or otherwise contacted with a pharmaceuticalcomposition of the invention, and the effect of such composition uponthe tissue sample is observed. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. In various specific embodiments, in vitroassays can be carried out with representative cells of cell typesinvolved in an autoimmune or inflammatory disorder (e.g., T cells), todetermine if a pharmaceutical composition of the invention has a desiredeffect upon such cell types.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary. Said aspectsinclude the temporal regime of administering the prophylactic and/ortherapeutic agents, and whether such agents are administered separatelyor as an admixture.

Preferred animal models for use in the methods of the invention are, forexample, transgenic mice expressing human FcγRs on mouse effector cells,e.g., any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety) can be used in thepresent invention. Transgenic mice for use in the methods of theinvention include, but are not limited to, mice carrying human FcγRIIIA;mice carrying human FcγRIIA; mice carrying human FcγRIIB and humanFcγRIIIA; mice carrying human FcγRIIB and human FcγRIIA. Preferably,mutations showing the highest levels of activity in the functionalassays described above will be tested for use in animal model studiesprior to use in humans. Sufficient quantities of antibodies may beprepared for use in animal models using methods described supra, forexample using mammalian expression systems and purification methodsdisclosed and exemplified herein.

Mouse xenograft models may be used for examining efficacy of mouseantibodies generated against a tumor specific target based on theaffinity and specificity of the epitope bing domains of the diabodymolecule of the invention and the ability of the diabody to elicit animmune response (Wu et al., 2001, Trends Cell Biol. 11: S2-9).Transgenic mice expressing human FcγRs on mouse effector cells areunique and are tailor-made animal models to test the efficacy of humanFc-FcγR interactions. Pairs of FcγRIIIA, FcγRIIIB and FcγRIIA transgenicmouse lines generated in the lab of Dr. Jeffrey Ravetch (Through alicensing agreement with Rockefeller U. and Sloan Kettering Cancercenter) can be used such as those listed in the Table 11 below. TABLE 11Mice Strains Strain Background Human FcR Nude/CD16A KO None Nude/CD16AKO FcγRIIIA Nude/CD16A KO FcγR IIA Nude/CD16A KO FcγR IIA and IIIANude/CD32B KO None Nude/CD32B KO FcγR IIB

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford L. J.and Wilder R. L., “Arthritis and Autoimmunity in Animals”, in Arthritisand Allied Conditions: A Textbook of Rheumatology, McCarty et al.(eds.),Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneous animalmodels of inflammatory arthritis and autoimmune rheumatic diseases canalso be used to assess the anti-inflammatory activity of the combinationtherapies of invention. The following are some assays provided asexamples, and not by limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford L. J. and Wilder R. L., “Arthritis and Autoimmunity inAnimals”, in Arthritis and Allied Conditions: A Textbook ofRheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993),incorporated herein by reference in its entirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra P. et al., “Carrageenan-Induced Arthritis in theRat,” Inflammation, 24(2): 141-155, (2000). Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter C. A. et al., “Carrageenan-Induced Edema in Hind Paw of the Ratas an Assay for Anti-inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547, (1962). This assay has been used as a primary in vivo screenfor the anti-inflammatory activity of most NSAIDs, and is consideredpredictive of human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

Additionally, animal models for inflammatory bowel disease can also beused to assess the efficacy of the combination therapies of invention(Kim et al., 1992, Scand. J. Gastroentrol. 27:529-537; Strober, 1985,Dig. Dis. Sci. 30(12 Suppl):3S-10S). Ulcerative cholitis and Crohn'sdisease are human inflammatory bowel diseases that can be induced inanimals. Sulfated polysaccharides including, but not limited toamylopectin, carrageen, amylopectin sulfate, and dextran sulfate orchemical irritants including but not limited to trinitrobenzenesulphonicacid (TNBS) and acetic acid can be administered to animals orally toinduce inflammatory bowel diseases.

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have been developed(Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999,Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in Relevanceof Tumor Models for Anticancer Drug Development (1999, eds. Fiebig andBurger); Contributions to Oncology (1999, Karger); The Nude Mouse inOncology Research (1991, eds. Boven and Winograd); and Anticancer DrugDevelopment Guide (1997 ed. Teicher), herein incorporated by referencein their entireties.

Preferred animal models for determining the therapeutic efficacy of themolecules of the invention are mouse xenograft models. Tumor cell linesthat can be used as a source for xenograft tumors include but are notlimited to, SKBR3 and MCF7 cells, which can be derived from patientswith breast adenocarcinoma. These cells have both erbB2 and prolactinreceptors. SKBR3 cells have been used routinely in the art as ADCC andxenograft tumor models. Alternatively, OVCAR3 cells derived from a humanovarian adenocarcinoma can be used as a source for xenograft tumors.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

6. EXAMPLES

6.1 Design and Characterization of Covalent Bispecific Diabodies

A monospecific covalent diabody and a bispecific covalent diabody wereconstructed to asses the recombinant production, purification andbinding characteristics of each. The affinity purified diabody moleculesthat were produced by the recombinant expression systems describedherein were found by SDS-PAGE and SEC analysis to consist of a singledimerc species. ELISA and SPR analysis further revealed that thecovalent bispecific diabody exhibited affinity for both target antigensand could bind both antigens simultaneously.

Materials and Methods:

Construction and Design of Polypeptide Molecules: Nucleic acidexpression vectors were designed to produce four polypeptide constructs,schematically represented in FIG. 2. Construct 1 (SEQ ID NO:9) comprisedthe VL domain of humanized 2B6 antibody, which recognizes FcγRIIB, andthe VH domain of humained 3G8 antibody, which recognizes FcγRIIIA.Construct 2 (SEQ ID NO:11) comprised the VL domain of Hu3G8 and the VHdomain of Hu2B6. Construct 3 (SEQ ID NO:12) comprised the VL domain ofHu3G8 and the VH domain of Hu3G8. Construct 4 (SEQ ID NO:13) comprisedthe VL domain of Hu2B6 and the VH domain of Hu2B6.

PCR and Expression Vector Construction: The coding sequences of the VLor VH domains were amplified from template DNA using forward and reverseprimers designed such that the intial PCR products would containoverlapping sequences, allowing overlapping PCR to generate the codingsequences of the desired polypeptide constructs.

Initial PCR amplification of template DNA: Approximately 35 ng oftemplate DNA, e.g. light chain and heavy chain of antibody of interest;1 ul of 10 mM forward and reverse primers; 2.5 ul of 10× pfuUltra buffer(Stratagene, Inc.); 1 ul of 10 mM dNTP; 1 ul of 2.5 units/ul of pfuUltraDNA polymerase (Stratagene, Inc.); and distilled water to 25 ul totalvolume were gently mixed in a microfuge tube and briefly spun in amicrocentrifuge to collect the reaction mixture at the bottom of thetube. PCR reactions were performed using GeneAmp PCR System 9700 (PEApplied Biosystem) and the following settings: 94° C., 2 minutes; 25cycles of 94° C., each 15 seconds; 58° C., 30 seconds; and 72° C., 1minute.

The VL of Hu2B6 was amplified from the light chain of Hu2B6 usingforward and reverse primers SEQ ID NO: 57 and SEQ ID NO:58,respectively. The VH of Hu2B6 was amplified from the heavy chain ofHu2B6 using forward and reverse primers SEQ ID NO: 59 and SEQ ID NO:60,respectively. The VL of Hu3G8 was amplified from the light chain ofHu3G8 using forward and reverse primers SEQ ID NO: 57 and SEQ ID NO:61,respectively. The VH of Hu3G8 was amplified from the heavy chain ofHu3G8 using forward and reverse primers SEQ ID NO: 62 and SEQ ID NO:63,respectively.

PCR products were electrophoresed on a 1% agarose gel for 30 minutes at120 volts. PCR products were cut from the gel and purified usingMinElute GEI Extraction Kit (Qiagen, Inc.).

Overlapping PCR: Intitial PCR products were combined as described belowand amplified using the same PCR conditions described for initialamplification of template DNA. Products of overlapping PCR were alsopurified as described supra.

The nucleic acid sequence encoding construct 1, SEQ ID NO:9 (shownschematically in FIG. 2), was amplified by combining the PCR products ofthe amplifications of VL Hu2B6 and VH Hu3G8, and forward and reverseprimers SEQ ID NO:57 and SEQ ID NO:63, respectively. The nucleic acidsequence encoding construct 2, SEQ ID NO:11 (shown schematically in FIG.2), was amplified by combining the PCR products of the amplifications ofVL Hu3G8 and VH Hu2B6, and forward and reverse primers SEQ ID NO:57 andSEQ ID NO:60, respectively. The nucleic acid sequence encoding construct3, SEQ ID NO:12 (shown schematically in FIG. 2), was amplified bycombining the PCR products of the amplifications of VL Hu3G8 and VHHu3G8, and forward and reverse primers SEQ ID NO:57 and SEQ ID NO:63,respectively. The nucleic acid sequence encoding construct 4, SEQ IDNO:13 (shown schematically in FIG. 2), was amplified by combining thePCR products of the amplifications of VL Hu2B6 and VH Hu2B6, and forwardand reverse primers SEQ ID NO:57 and SEQ ID NO:60, respectively.

The forward primers of the VL domains (i.e., SEQ ID NO:57) and reverseprimers of the VH domains (i.e., SEQ ID NO:60 and SEQ ID NO:63)contained unique restriction sites to allow cloning of the final productinto an expression vector. Purified overlapping PCR products weredigested with restriction endonucleases Nhe I and EcoR I, and clonedinto the pCIneo mammalian expression vector (Promega, Inc.). Theplasmids encoding constructs were designated as identified in Table 12:TABLE 12 PLASMID CONSTRUCTS Encoding Construct Plasmid DesignationInsert 1 pMGX0669 hu2B6VL-hu3G8VH 2 pMGX0667 hu3G8VL-hu2B6VH 3 pMGX0666hu3G8VL-hu3G8VH 4 pMGX0668 hu2B6VL-hu2B6VH

Polypeptide/diabody Expression: pMGX0669, encoding construct 1, wascotransfected with pMGX0667, encoding construct 2, in HEK-293 cellsusing Lipofectamine 2000 according to the manufacturer's directions(Invitrogen). Co-transfection of these two plasmids was designed to leadto the expression of a covalent bispecific diabody (CBD) immunospecificfor both FcγRIIB and FcγRIIIA (the h2B6-h3G8 diabody). pMGX0666 andpMGX0668, encoding constructs 3 and 4, respectively, were separatelytransfected into HEK-293 cells for expression of a covalent monospecificdiabody (CMD), immunospecific for FcγRIIIA (h3G8 diabody) and FcγRIIB(h2B6 diabody), respectively. Following three days in culture, secretedproducts were purified from the conditioned media.

Purification: Diabodies were captured from the conditioned medium usingthe relevant antigens coupled to CNBr activated Sepharose 4B. Theaffinity Sepharose resin was equilibrated in 20 mM Tris/HCI, pH 8.0prior to loading. After loading, the resin was washed with equilibrationbuffer prior to elution. Diabodies were eluted from the washed resinusing 50 mM Glycine pH 3.0. Eluted diabodies were immediatelyneutralized with 1M Tris/HCl pH 8.0 and concentrated using acentrifugation type concentrator. The concentrated diabodies werefurther purified by size exclusion chromatography using a Superdex 200column equilibrated in PBS.

SEC: Size exclusion chromatography was used to analyze the approximatesize and heterogeneity of the diabodies eluted from the column. SECanalysis was performed on a GE healthcare Superdex 200 HR 10/30 columnequilibrated with PBS. Comparison with the elution profiles of a fulllength IgG (˜150 kDa), an Fab fragment (˜50 kDa) and a single chain Fv(˜30 kDa) were used as controls).

ELISA: The binding of eluted and purified diabodies was characterized byELISA assay, as described in 5.4.2. 50 ul/well of a 2 ug/ml solution ofsCD32B-Ig was coated on 96-well Maxisorp plate in Carbonate buffer at 4°C. over night. The plate was washed three times with PBS-T (PBS, 0.1%Tween 20) and blocked by 0.5% BSA in PBS-T for 30 minutes at roomtemperature. Subsequently, h2B6-h3G8 CBD, h2B6 CMD, or h3G8 CMD werediluted into the blocking buffer in a serial of two-fold dilutions togenerate a range of diabody concentrations, from 0.5 μg/ml to 0.001μg/ml. The plate was then incubated at room temperature for 1 hour.After washing with PBS-T three times, 50 ul/well of 0.2 ug/mlsCD16A-Biotin was added to each well. The plate was again incubated atroom temperature for 1 hour. After washing with PBS-T three times, 50ul/well of a 1:5000 dilution of HRP conjugated streptavidin (AmershamPharmacia Biotech) was used for detection. The HRP-streptavidin wasallowed to incubate for 45 minutes at room temperature. The plate waswashed with PBS-T three times and developed using 80 ul/well of TMBsubstrate. After a 10 minute incubation, the HRP-TMB reaction wasstopped by adding 40 ul/well of 1% H₂SO₄. The OD450 nm was read by usinga 96-well plate reader and SOFTmax software, and results plotted usingGraphPadPrism 3.03 software.

BIAcore Assay: The kinetic parameters of the binding of eluted andpurified diabodies were analyzed using a BIAcore assay (BIAcoreinstrument 1000, BIAcore Inc., Piscataway, N.J.) and associated softwareas described in section 5.4.3.

sCD16A, sCD32B or sCD32A (negative control) were immobilized on one ofthe four flow cells (flow cell 2) of a sensor chip surface through aminecoupling chemistry (by modification of carboxymethyl groups with mixtureof NHS/EDC) such that about 1000 response units (RU) of either receptorwas immobilized on the surface. Following this, the unreacted activeesters were “capped off” with an injection of 1M Et-NH2. Once a suitablesurface was prepared, covalent bispecific diabodies (h2B6-h3G8 CBD) orcovalent monospecific diabodies (h2B6 CMD or h3G8 CMB) were passed overthe surface by 180 second injections of a 6.25-200 nM solution at a 70mL/min flow rate. h3G8 scFV was also tested for comparison.

Once an entire data set was collected, the resulting binding curves wereglobally fitted using computer algorithms supplied by the manufacturer,BIAcore, Inc. (Piscataway, N.J.). These algorithms calculate both theK_(on) and K_(off), from which the apparent equilibrium bindingconstant, K_(D) is deduced as the ratio of the two rate constants (i.e.,K_(off)/K_(on)). More detailed treatments of how the individual rateconstants are derived can be found in the BIAevaluaion Software Handbook(BIAcore, Inc., Piscataway, N.J.).

Association and dissociation phases were fitted separately. Dissociationrate constant was obtained for interval 32-34 sec of the 180 secdissociation phase; association phase fit was obtained by a 1:1 Langmuirmodel and base fit was selected on the basis R_(max) and chi² criteriafor the bispecific diabodies and scFv; Bivalent analyte fit was used forCMD binding.

Results

SDS-PAGE analysis under non-reducing conditions revealed that thepurified product of the h3G8 CMD, h2B6 CMD and h2B6-h3G8 CBD expressionsystems were each a single species with an estimated molecular weight ofapproximately 50 kDa (FIG. 3, lanes 4, 5 and 6, respectively). Underreducing conditions, the product purified from either of the CMDexpression systems ran as a single band (lanes 1 and 2), while theproduct purified from the h2B6-h3G8 CBD system was revealed to be 2separate proteins (FIG. 3, lane 3). All polypeptides purified from theexpression system and visualized by SDS-PAGE under reducing conditionsmigrated at approximately 28 kDa.

SEC analysis of each of the expression system products also revealed asingle molecular species (FIG. 4B), each of which eluted at the sameapproximate time as an Fab fragment of IgG (˜50 kDa) (FIG. 4A). Theresults indicate that affinity purified product was a homogenouscovalent homodimer for the case of CMD expression system and ahomogenous covalent heterodimer for the case of the h2B6-h3G8 CBD.

An ELISA sandwich assay was used to test binding of the h2B6-h3G8 CBDfor specificity to either or both of CD32B and/or CD16A (FIG. 5). CD32Bserved as the target antigen and CD16A was used as the secondary probe.The positive signal in the ELIZA revealed that the heterodimerich2B6-h3G8 CBD had specificity for both antigens. Similar testing of theh3G8 CMD (which should not bind CD32B) showed no signal.

SPR analysis indicated that h3G8 CMD immunospecifically recognized sCD16but not sCD32B, that h2B6 CMD immunospecifically recognized sCD32B butnot sCD16, and that h2B6-h3G8 CBD immunospecifically recognized bothsCD16 and sCD32B (FIGS. 6A-B). None of the diabodies tested bound thecontrol receptor, sCD32A (FIG. 6C).

SPR analysis was also used to estimate the kinetic and equilibriumconstants of the CMDs and h2B6-h3G8 CBD to sCD16 and/or sCD32B. Resultswere compared to the same constants calculated for an h3G8 scFV. FIGS.7A-E show the graphical results of the SPR analysis. The kinetic on andoff rates, as well as the equilibrium constant, calculated from theresults depicted in FIG. 7 are provided in Table 13. TABLE 13 Kineticand Equilibrium Constants Calculated from BIAcore Data. Receptor/Analytek-on k-off Kd sCD16/h3G8 diabody 2.3 × 10⁵ 0.004 18.0 sCD16/h2B6-h3G8CBD 4.6 × 10⁵ 0.010 22.7 sCD16/h3G8 scFv 3.2 × 10⁵ 0.013 38.7sCD32B/h2B6-h3G8 CBD 3.6 × 10⁵ 0.005 15.0 sCD32B/h2B6 diabody 6.2 × 10⁵0.013 21.0

Coupled with the results of the ELISA analysis, the studies confirm thatthe h2B6-h3G8 covalent heterodimer retained specificity for both CD32Band CD16, and was capable of binding both antigens simultaneously. Themolecule is schematically represented in FIG. 8.

6.2 Design and Characterization of Covalent Bispecific DiabodiesComprising Fc Domains

In an effort to create an IgG like molecule, i.e., comprising an Fcdomain, one of the polypeptides comprising the heterodimeric CBDmolecule presented in Example 6.1 was modified to further comprise an Fcdomain (creating a ‘heavier’ and ‘lighter’ chain, analogous to anantibody heavy and light chain). The heterodimeric bispecific moleculewould then contain an Fc domain that will dimerize with a homologousmolecule, forming a tetrameric IgG-like molecule with tetravalency (i.e,formed by dimerization via the Fc domains of the heterodimericbispecific molecules). Interestingly, such tetrameric molecules were notdetected in the conditioned media of recombinant expression systemsusing functional assays, e.g., testing the conditioned media forimmunospecific binding to target antigens. Instead, only a dimericmolecule, comprising monomers consisting of a VL, VH and Fc domain, weredetected in such functional assays. To test whether stability of thetheoretical tetrameric structure was at issue, polypeptides comprisingthe Fc domain were engineered to further comprise a hinge region whilethe polypeptides comprising the ‘lighter’ chain were engineered tofurther comprise the 6 C-terminal amino acids of the constant domain ofthe human kappa light chain. When such reengineered ‘heavier’ and‘lighter; chains were co-expressed in the recombinant expressionsystems, functional assays detected diabody molecules that were able toimmunospecifically bind both of the target antigens and anti-Fcantibodies.

Materials and Methods

Construction and Design of Polypeptide Molecules: Nucleic acidexpression vectors were designed to produce modified versions ofconstructs 1 and 2 presented in Example 6.1. Construct 5 (SEQ ID NO: 14)and 6 (SEQ ID NO:15), were created by engineering construct 1 and 2,respectively to further comprise an Fc domain. Construct 7 (SEQ ID NO:16) was created by engineering construct 1 was to further comprise thesequence FNRGEC (SEQ ID NO: 23) at its C-terminus. Construct 8 (SEQ IDNO:18) was created by engineering construct 2 to further comprise ahinge region and Fc domain (comprising V215A mutation). Schematicrepresentation of constructs 5-8 is shown in FIG. 9.

PCR and Expression Vector Construction: All PCR and PCR productpurification protocols were as described in Example 6.1 PlasmidspMGXO669 and pMGXO667 served as templates for the coding sequences ofconstructs 1 and 2, respectively. The coding sequences for the of HuIgGFc domain and/or hinge domain were SEQ ID NO:5 or SEQ ID NO:1 and SEQ IDNO:5, respectively. The coding sequences of the template DNAs wereamplified using forward and reverse primers such that the PCR productswould contain overlapping sequences, allowing overlapping PCR togenerate the coding sequences of the desired products.

The coding sequence of construct 1 was amplified from pMGXO669 usingforward and reverse primers SEQ ID NO:57 and SEQ ID NO:64, respectively.The coding sequence of construct 2 was amplified from pMGX0667 usingforward and reverse primers SEQ ID NO:57 and SEQ ID NO:65, respectively.HuIgG hinge-Fc was amplified using forward and reverse primers SEQ IDNO:67 and SEQ ID NO:68, respectively. Construct 7 (SEQ ID NO:16) wasamplified from pMGX0669 using forward and reverse primers SEQ ID NO:57and SEQ ID NO:69.

Overlapping PCR: Initial PCR products were combined as described below,amplified and purified as described in example 6.1.

The nucleic acid sequence encoding construct 5, SEQ ID NO:14 (shownschematically in FIG. 9), was amplified by combining the PCR products ofthe amplifications of construct 1 and HuIgG Fc, and forward and reverseprimers SEQ ID NO:57 and SEQ ID NO:66, respectively. The nucleic acidsequence encoding construct 6, SEQ ID NO:15 (shown schematically in FIG.9), was amplified by combining the PCR products of the amplifications ofconstruct 2 and HuIgG Fc, and forward and reverse primers SEQ ID NO:57and SEQ ID NO:66, respectively. The nucleic acid sequence encodingconstruct 8, SEQ ID NO:18 (shown schematically in FIG. 9), was amplifiedby combining the PCR products of the amplifications of construct 2 andHuIgG hinge-Fc, and forward and reverse primers SEQ ID NO:57 and SEQ IDNO:68, respectively.

Final products were cloned into pCIneo mammalian expression vector(Promega, Inc.) as previously described. The plasmid encoding constructswere designated as identified in Table 14: TABLE 14 PLASMID CONSTRUCTSEncoding Construct Plasmid Designation Insert 5 pMGX0676hu2B6VL-hu3G8VH-huFc 6 pMGX0674 hu3G8VL-hu2B6VH-huFc 7 pMGX0677Hu2B6VL-hu3G8VH- FNRGEC 8 pMGX0678 Hu3G8VL-hu2B6VH-huhinge- Fc (A215V)

Polypeptide/diabody Expression: Four separate cotransfections into inHEK-293 cells using Lipofectamine 2000, as described in section 6.1,were performed: pMGX0669 and pMGX0674, encoding constructs 1 and 6,respectively;; pMGX0667 and pMGX0676, encoding constructs 2 and 5,respectively; and pMGX0677 and pMGX0678, encoding constructs 7 and 8,respectively.

Co-transfection of these plasmids was designed to lead to the expressionof a bispecific diabody (CBD) of tetravalency with IgG-like structure,immuno specific for both FcγRIIB and FcγRIIIA. An additionalcotransfection was also performed: pMGX0674 and pMGX0676, encodingconstructs 6 and 5, respectively. Following three days in culture,conditioned media was harvested. The amount of secreted product in theconditioned media was quantitiated by anti IgG Fc ELISA using purifiedFc as a standard. The concentrations of product in the samples was thennormalized based on the quantitation, and the normalized samples usedfor the remaining assays.

ELISA: The binding of diabody molecules secreted into the medium wasassayed by sandwich ELISA as described, supra. Unless indicated, CD32Bwas used to coat the plate, i.e., as the target protein, andHRP-conjugated CD16 was used as the probe.

Results

An ELISA assay was used to test the normalized samples from therecombinant expression systems comprising constructs 1 and 6(pMGX669-pMGX674), constructs 2 and 5 (pMGX667-pNGX676) and constructs 5and 6 (pMGX674-pMGX676) for expression of diabody molecules capable ofsimultaneous binding to CD32B and CD16A (FIG. 10). The ELISA dataindicated that co-transfection with constructs 1 and 6 orco-transfection with constructs 2 and 5 failed to produce a product thatcould bind either or both antigens (FIG. 10, ▭ and ▴, respectively).However, co-transfection of constructs 5 and 6 lead to secretion of aproduct capable of binding to both CD32B and CD16 antigens. The latterproduct was a dimer of constructs 5 and 6, containing one binding sitefor each antigen with a structure schematically depicted in FIG. 11.

In order to drive formation of an IgG like heterotetrameric structure,the coding sequence for six additional amino acids was appended to theC-terminal of construct 1, generating construct 7 (SEQ ID NO:16 andshown schematically in FIG. 9). The six additional amino acids, FNRGEC(SEQ ID NO:23), were derived from the C-terminal end of the the Kappalight chain and normally interact with the upper hinge domain of theheavy chain in an IgG molecule. A hinge domain was then engineered intoconstruct 6, generating construct 8 (SEQ ID NO:18 and FIG. 9). Construct8 additionally comprised an amino-acid mutation in the upper hingeregion, A215V. Expression plasmids encoding construct 7 and construct 8,pMGX677 and pMGX678, respectively, were then cotransfected into HEK-293cells and expressed as described.

Diabody molecules produced from the recombinant expression systemcomprising constructs 7 and 8 (pMGX0677+pMGX0678), were compared in anELISA assay for binding to CD32B and CD16A to diabody molecules producedfrom expression systems comprising constructs 1 and 6 (pMGX669+pMGX674),constructs 2 and 8 (pMGX669+pMGX678), and constructs 6 and 7(pMGX677+pMGX674) (FIG. 12).

As before, the molecule produced by the expression system comprisingconstructs 1 and 6 (pMGX669+pMGX674) proved unable to bind both CD32Aand CD16A (FIG. 10 and FIG. 12). In contrast, the product from theco-expression of either constructs 7 and 6 (pMGX0677+pMGX0674) or fromthe co-expression of constructs 7 and 8 (pMGX0677-pMGX0678) were able tobind both CD32B and CD16 (FIG. 12). It is noted that construct 7 isanalogous to construct 1, with the exception that construct 7 comprisesthe C-terminal sequence FNRGEC (SEC ID NO:23); and that construct 8 isanalogous to construct 6, except that construct 8 comprises a hingedomain and the mutation A215V. The data indicate that the addition ofthe 6 extra amino-acids from the C-terminus of the C-kappa light chain(FNRGEC; SEQ ID NO:23) to the non-Fc bearing, ‘lighter,’ chain helpedstabilize the formation of the tetrameric IgG-like diabody molecules,regardless of whether the corresponding heavier chain comprised a hingedomain (i.e., pMGX0677+pMGX0674 and pMGX0677-pMGX0678, FIG. 12). Theaddition of the hinge domain to the Fc bearing ‘heavier’ polypeptide,without the addition of the FNRGEC (SEQ ID NO:23) C-terminal sequence tothe corresponding ‘lighter’ chain, was apparently unable to effectsimilar stabilization (i.e., lack of binding by product ofco-transfection of constructs 2 and 8 (pMGX669+pMGX678)). The structureof the tetrameric diabody molecule is schematically represented in FIG.13.

6.3 Effect of Domain Order and Additional Disulfide Bonds on Formationof Tetremeric IgG-Like Diabody

The effect of additional stabilization between the ‘lighter’ and‘heavier’ polypeptide chains of the tetrameric IgG-like diabody moleculewas investigated by substitution of selected residues on the polypeptidechains with cysteines. The additional cysteine residues provide foradditional disulfide bonds between the ‘heavier’ and ‘lighter’ chains.Additionally, domain order on binding activity was investigated bymoving the Fc domain or the hinge-Fc domain from the C-terminal end ofthe polypeptide chain to the N-terminus. Although the binding activityof the molecule comprising the additional disulfide bonds was notaltered relative to earlier constructed diabody molecules with suchbonds, transferring the Fc or hinge-Fc domain to the N-terminus of the‘heavier’ polypeptide chain comprising the diabody surprisingly improvedbinding affinity and/or avidity of the bispecific molecule to one orboth of its target antigens.

Materials and Methods

Construction and Design of Polypeptide Molecules: Nucleic acidexpression vectors were designed to produce modified versions ofconstructs 5, 6 and 8 presented in Example 6.2. Construct 9 (SEQ IDNO:19) and construct 10 (SEQ ID NO:20) (both shown schematically in FIG.13) were analogous to constructs 8 and 6, with the exception that Fcdomain or hinge-Fc domain, respectively, was shifted from the C-terminusof the polypeptide to the N-terminus. Additionally all Fc domains usedwere wild-type IgG1 Fc domains. Construct 11, SEQ ID NO:21, (shownschematically in FIG. 14) was analogous to construct 2 from Example 6.1except that the C-terminus was designed to further comprise the sequenceFNRGEC (SEQ ID NO:23). Construct 12, SEQ ID NO:22 (shown schematicallyin FIG. 14) was analogous to construct 5 from Example 6.2 except thatthe Fc domain further comprised a hinge region. Also, for constructs 11and 12, the 2B6 VL domain and 2B6 VH domain comprised a single aminoacid modification (G105C and G44C, respectively) such that a glycine ineach domain was replaced by cysteine.

PCR and Expression Vector Construction: All PCR and PCR productpurification protocols were as described in Example 6.1 and 6.2

Overlapping PCR: Final products were constructed, amplified and purifiedusing methods described in example 6.1 and example 6.2.

Final products were cloned into pCIneo mammalian expression vector(Promega, Inc.) as previously described. The plasmid encoding constructswere designated as identified in Table 15: TABLE 15 PLASMID CONSTRUCTSEncoding Construct Plasmid Designation Insert 9 pMGX0719Huhinge/Fc-hu3G8VL- hu2B6VH 10 pMGX0718 HuFc-hu2B6VL-hu3G8VH 11 pMGX0716Hu2B6VL(G/C)-hu3G8VH- huhingeFC 12 pMGX0717 Hu3G8VL-hu2B6VH (G/C)-FNRGEC

Polypeptide/diabody Expression: Three separate cotransfections in to inHEK-293 cells using Lipofectamine 2000, as described in section 6.1,were performed: pMGX0669 and pMGX0719, encoding constructs 1 and 9,respectively; pMGX0669 and pMGX0718, encoding constructs 1 and 10,respectively; and pMGX0617 and pMGX0717, encoding constructs 11 and 12,respectively. Co-transfection of these plasmids was designed to lead tothe expression of a bispecific diabody (CBD) of tetravalency withIgG-like structure, immunospecific for both FcγRIIB and FcγRIIIA.Following three days in culture, conditioned media was harvested. Theamount of secreted product in the conditioned media was quantitiated byanti IgG Fc ELISA using purified Fc as a standard. The concentrations ofproduct in the samples was then normalized based on the quantitation,and the normalized samples used for the remaining assays.

ELISA: The binding of diabody molecules secreted into the medium wasassayed by sandwich ELISA as described, supra. Unless indicated, CD32Bwas used to coat the plate, i.e., as the target protein, andHRP-conjugated CD16 was used as the probe.

Western Blot: Approximately 15 ml of conditioned medium form the threeabove-described cotransfections were analyzed by SDS-PAGE undernon-reducing conditions. One gel was stained with Simply Blue Safestain(Invitrogen) and an identical gel was transferred to PVDF membrane(Invitrogen) using standard transfer methods. After transfer, themembrane was blocked with 5% dry skim milk in IX PBS. The membrane wasthen incubated in 10 ml of 1:8,000 diluted HRP conjugated Goat antihuman IgG1 H+L in 2% dry skim milk 1XPBS/0.1% Tween 20 at roomtemperature for 1 hr with gentle agitation. Following a wash with 1XPBS/0.3% Tween 20, 2× 5 min each, then 20 min at room temperature, themembrane was developed with ECL Western blotting detection system(Amersham Biosciences) according to the manufacturer's instructions. Thefilm was developed in X-ray processor.

Results

Conditioned media from the recombinant expression systems comprisingconstructs 1 and 9; constructs 1 and 10; and constructs 11 and 12 wereanalyzed by SDS-PAGE (under non reducing conditions) analysis andWestern-blotting (using an anti-IgG as the probe). Western blot revealedthat the product from the systems comprising constructs 11 and 12 orcomprising constructs 9 and 1 predominately formed a single species ofmolecule of approximately 150 kDa (FIG. 14, lanes 3 and 2,respectively). Both of these products have engineered internal disulfidebonds between the ‘lighter’ and ‘heavier’ chains comprising the diabody.In contrast, the molecule without engineered internal disulfide bondsbetween the ‘lighter’ and ‘heavier’ chains, formed of constructs 10 and1, formed at least two molecular species of molecular weights ˜75 and˜100 kDa (FIG. 14, lane 1).

Despite the results of the Western Blot, each of the three products wasfound capable of binding both CD32A and CD16 (FIG. 15). Surprisingly,relative to the product comprising a C-terminal hinge-Fc domain (formedof constructs 11 and 12), the product from both systems wherein the Fc(or Fc-hinge) domain was at the amino terminus of the Fc containingpolypeptide chain (i.e., the ‘heavier’ chain) (constructs 9+1 andconstructs 10+1) demonstrated enhanced affinity and/or avidity to one orboth of its target peptides (i.e. CD32B and/or CD16).

6.4 Effect of Internal/External Cleavage Site on Processing ofPolyprotein Precursor and Expression of Covalent Bispecific Diabody;Design and Characterization of Bispecific Diabody Comprising Portions ofHuman IgG Lambda Chain and Hinge Domain

As described herein, the individual polypeptide chains of the diabody ordiabody molecule of the invention may be expressed as a singlepolyprotein precursor molecule. The ability of the recombinant systemsdescribed in Examples 6.1-6.3 to properly process and express afunctional CBD from such a polyprotein precursor was tested byengineering a nucleic acid to encode, both the first and secondpolypeptide chains of a CBD separated by an internal cleavage site, inparticular, a furin cleavage site. Functional, CBD was isolated from therecombinant system comprising the polyprotein precursor molecule.

As discussed in Example 6.3, addition of the 6 C-terminal amino acidsfrom the human kappa light chain, FNRGEC (SEQ ID NO:23), was found tostabilize diabody formation—presumably through enhanced inter-chaininteraction between the domains comprising SEQ ID NO:23 and thosedomains comprising an Fc domain or a hinge-Fc domain. The stabilizingeffect of this lambda chain/Fc like interaction was tested in CBDwherein neither polypeptide chain comprised an Fc domain. Onepolypeptide chain of the diabody was engineered to comprise SEQ ID NO:23at its C-terminus; the partner polypeptide chain was engineered tocomprise the amino acid sequence VEPKSC (SEQ ID NO:79), which wasderived from the hinge domain of an IgG. Comparison of this CBD to thatcomprised of constructs 1 and 2 (from example 6.1) revealed that the CBDcomprising the domains derived from hinge domain and lambda chainexhibited slightly greater affinity to one or both of its targetepitopes.

Materials and Methods

-   Construction and Design of Polypeptide Molecules: Polyprotein    precursor: Nucleic acid expression vectors were designed to produce    2 poyprotein precursor molecules, both represented chematically in    FIG. 17. Construct 13 (SEQ ID NO:97) comprised from the N-terminus    of the polypeptide chain, the VL domain of 3G8, the VH domain of    2.4G2 (which binds mCD32B), a furin cleavage site, the VL domain of    2.4G2 and the VH domain of 3G8. The nucleotide sequence encoding    construct 13 is provided in SEQ ID NO:98. Construct 14 (SEQ ID    NO:99) (FIG. 17), comprised from the N-terminus of the polypeptide    chain, the VL domain of 3G8, the VH domain of 2.4G2 (which binds    mCD32B), a furin cleavage site, a FMD (Foot and Mouth Disease Virus    Protease C3) site, the VL domain of 2.4G2 and the VH domain of 3G8.    The nucleotide sequence encoding construct 14 is provided in SEQ ID    NO:100.

Nucleic acid expression vectors were designed to produce modifiedversions of constructs 1 and 2 presented in Example 6.1. Construct 15(SEQ ID NO:101) (FIG. 17) was analagous to construct 1 (SEQ ID NO:9),presented in example 6.1, with the exception that the C-terminus ofcontruct 15 comprised the amino acid sequence FNRGEC (SEQ ID NO:23). Thenucleic acid sequence encoding construct 15 is provided in SEQ IDNO:102. Construct 16 (SEQ ID NO:103) (FIG. 17) was analogous toconstruct 2, presented in Example 6.1, with the exception that theC-terminus of construct 16 comprised the amino acid sequence VEPSK (SEQID NO:79). The nucleic acid sequence encoding construct 16 is providedin SEQ ID NO:104.

PCR and Expression Vector Construction: All PCR and PCR productpurification protocols were as described in Example 6.1 and 6.2

Overlapping PCR: Final products were constructed, amplified and purifiedusing methods described in example 6.1 and example 6.2 with appropriateprimers

Final products were cloned into pCIneo mammalian expression vector(Promega, Inc.) as previously described. The plasmid encoding constructswere designated as identified in Table 16: TABLE 16 PLASMID CONSTRUCTSEncoding Construct Plasmid Designation Insert 13 pMGX07503G8VL-2.4G2VH-Furin- 2.4G2VL-3G8VH 15 pMGX0752 Hu2B6VL-Hu3G8VH- FNRGEC16 pMGX0753 Hu3G8VL-Hu2B6VH- VEPKSC

Polypeptide/diabody Expression: One transfection and one cotransfectioninto in HEK-293 cells using Lipofectamine 2000, as described in section6.1, were performed: single: pMGX0750, encoding construct 13; andcotranfection: pMGX0752 and pMGX0753, encoding constructs 15 and 16,respectively. Following three days in culture, conditioned media washarvested, and secreted product affinity purified as described.

ELISA: The binding of diabody molecules secreted into the medium wasassayed by sandwich ELISA as described, supra. Murine CD32B was used tocoat the plate, i.e., as the target protein, and HRP-conjugated CD16Awas used as the probe for the product of the co-transfection ofconstructs 15 and 16. mCD32B was used as the target protein andbiotin-conjugated CD16A was used as the probe for the recombinant systemcomprising construct 13.

Results

Conditioned media from the recombinant expression systems comprisingconstructs 13 was analysed by sandwich ELISA. The ELISA assay tested thebinding of the CBD for specificity to either or both of mCD32B and/orCD16 (FIG. 18). CD32B served as the target antigen and CD16A was used asthe secondary probe. The positive signal in the ELISA revealed that theheterodimeric h2.4G2-h3G8 CBD produced from the polyprotein precursorhad specificity for both antigens.

Similarly, the purified product generated by cotransfection of thevectors encoding constructs 15 and 16 was tested in an ELISA assay andcompared to the product comprised of contructs 1 and 2 (Example 6.1).CD32B served as the target antigen and CD16A was used as the secondaryprobe. As with the product comprised of constructs 1 and 2, the productof constructs 15 and 16 was found to be capable of simultaneouslybinding CD32B and CD16A. In fact, the product of constructs 15 and 16showed slightly enhanced affinity for one or both of the targetantigens, i.e. CD32B or CD16A. This is perhaps due to increasedstability and or fidelity (relative to a wild type VH-VL domaininteraction) of the interchain association afforded by the interactionof the lambda chain region, FNRGEC (SEQ ID NO:23) and hinge regionVEPKSC (SEQ ID NO:79), which is absent in the product comprised ofconstructs 1 and 2.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled. Such modifications areintended to fall within the scope of the appended claims.

All references, patent and non-patent, cited herein are incorporatedherein by reference in their entireties and for all purposes to the sameextent as if each individual publication or patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety for all purposes.

1. A diabody molecule comprising a first polypeptide chain and a secondpolypeptide chain, which first polypeptide chain comprises (i) a firstdomain comprising a binding region of a light chain variable domain of afirst immunoglobulin (VL1) specific for a first epitope, (ii) a seconddomain comprising a binding region of a heavy chain variable domain of asecond immunoglobulin (VH2) specific for a second epitope, and (iii) athird domain comprising an Fc domain or portion thereof, which firstdomain and second domain are covalently linked such that the firstdomain and second domain do not associate to form an epitope bindingsite; which second polypeptide chain comprises (i) a fourth domaincomprising a binding region of a light chain variable domain of thesecond immunoglobulin (VL2), (ii) a fifth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), and (iii) a sixth domain comprising an Fc domain, which fourthdomain and fifth domain are covalently linked such that the fourthdomain and fifth domain do not associate to form an epitope bindingsite; wherein the first domain and the fifth domain associate to form afirst binding site (VL1)(VH1) that binds the first epitope; and whereinthe second domain and the fourth domain associate to form a secondbinding site (VL2)(VH2) that binds the second epitope.
 2. The diabodymolecule of claim 1, wherein the third domain further comprises a hingedomain.
 3. A diabody molecule comprising a first polypeptide chain and asecond polypeptide chain, which first polypeptide chain comprises (i) afirst domain comprising a binding region of a light chain variabledomain of a first immunoglobulin (VL1) specific for a first epitope,(ii) a second domain comprising a binding region of a heavy chainvariable domain of a second immunoglobulin (VH2) specific for a secondepitope, and (iii) a third domain comprising a hinge domain and an Fcdomain or portion thereof, which first domain and second domain arecovalently linked such that the first domain and second domain do notassociate to form an epitope binding site; which second polypeptidechain comprises (i) a fourth domain comprising a binding region of alight chain variable domain of the second immunoglobulin (VL2), (ii) afifth domain comprising a binding region of a heavy chain variabledomain of the first immunoglobulin (VH1), and (iii) a sixth domaincomprising the amino acid sequence of at least the C-terminal 2 to 8amino acid residues of a human light chain constant domain, which fourthdomain and fifth domain are covalently linked such that the fourthdomain and fifth domain do not associate to form an epitope bindingsite; wherein the first domain and the fifth domain associate to form afirst binding site (VL1)(VH1) that binds the first epitope; and whereinthe second domain and the fourth domain associate to form a secondbinding site (VL2)(VH2) that binds the second epitope.
 4. A diabodymolecule comprising a first polypeptide chain and a second polypeptidechain, which first polypeptide chain comprises (i) a first domaincomprising a binding region of a light chain variable domain of a firstimmunoglobulin (VL1) specific for a first epitope, (ii) a second domaincomprising a binding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope, and (iii) a thirddomain comprising a hinge domain, which first domain and second domainare covalently linked such that the first domain and second domain donot associate to form an epitope binding site; which second polypeptidechain comprises (i) a fourth domain comprising a binding region of alight chain variable domain of the second immunoglobulin (VL2), (ii) afifth domain comprising a binding region of a heavy chain variabledomain of the first immunoglobulin (VH1), and (iii) a sixth domaincomprising the amino acid sequence of at least the C-terminal 2 to 8amino acid residues of a human light chain constant domain, which fourthdomain and fifth domain are covalently linked such that the fourthdomain and fifth domain do not associate to form an epitope bindingsite; wherein the first domain and the fifth domain associate to form afirst binding site (VL1)(VH1) that binds the first epitope; and whereinthe second domain and the fourth domain associate to form a secondbinding site (VL2)(VH2) that binds the second epitope.
 5. The diabodymolecule of claim 3 or 4, wherein the human light chain is a human kappalight chain.
 6. The diabody molecule of claim 3 or 4 wherein the humanlight chain is a lambda light chain.
 7. The diabody molecule of claim 5,wherein the sixth domain comprises the amino acid sequence of theC-terminal 6 amino acids, which sequence is PheAsnArgGlyGluCys (SEQ IDNO:23).
 8. The diabody molecule of claim 1, 2, 3 or 4, wherein the firstpolypeptide chain and the second polypeptide chain are covalently linkedvia at least one disulfide bond between at least one cysteine residueoutside of the first domain and the second domain on the firstpolypeptide chain and at least one cysteine residue outside of thefourth domain and the fifth domain on the second polypeptide chain. 9.(canceled)
 10. A diabody molecule comprising a first polypeptide chainand a second polypeptide chain, which first polypeptide chain comprises(i) a first domain comprising a binding region of a light chain variabledomain of a first immunoglobulin (VL1) specific for a first epitope,(ii) a second domain comprising a binding region of a heavy chainvariable domain of a second immunoglobulin (VH2) specific for a secondepitope, and (iii) a third domain comprising an Fc domain or portionthereof, which first domain and second domain are covalently linked suchthat the first domain and second domain do not associate to form anepitope binding site; which second polypeptide chain comprises (i) afourth domain comprising a binding region of a light chain variabledomain of the second immunoglobulin (VL2), and (ii) a fifth domaincomprising a binding region of a heavy chain variable domain of thefirst immunoglobulin (VH1), which fourth domain and fifth domain arecovalently linked such that the fourth and fifth domains do notassociate to form an epitope binding site; wherein the first domain andthe fifth domain associate to form a first binding site (VL1)(VH1) thatbinds the first epitope; wherein the second domain and the fourth domainassociate to form a second binding site (VL2)(VH2) that binds the secondepitope; and wherein the third domain is N-terminal to both the firstdomain and the second domain. 11-23. (canceled)
 24. A diabody moleculecomprising a first and a second polypeptide chain, which firstpolypeptide chain comprises (i) a first domain comprising a bindingregion of a light chain variable domain of a first immunoglobulin (VL1)specific for a first epitope, and (ii) a second domain comprising abinding region of a heavy chain variable domain of a secondimmunoglobulin (VH2) specific for a second epitope, which first domainand second domain are covalently linked such that the first domain andsecond domain do not associate to form an epitope binding site; whichsecond polypeptide chain comprises (i) a third domain comprising abinding region of a light chain variable domain of the secondimmunoglobulin (VL2), and (ii) a fourth domain comprising a bindingregion of a heavy chain variable domain of the first immunoglobulin(VH1), which third domain and fourth domain are covalently linked suchthat the third domain and fourth domain do not associate to form anepitope binding site; wherein the first domain and the third domainassociate to form a first binding site (VL1)(VH1) that binds the firstepitope, which epitope binding site is specific for CD32B; wherein thesecond domain and the fourth domain associate to form a second bindingsite (VL2)(VH2) that binds the second epitope, which epitope bindingsite is specific for CD16; and wherein the first polypeptide chain andthe second polypeptide chain are covalently linked via a disulfide bondbetween at least one cysteine residue outside of the first domain andthe second domain on the first polypeptide chain and at least onecysteine residue outside of the third domain and the fourth domain onthe second polypeptide chain, which cysteine residue on the firstpolypeptide chain is not at the C-terminus of the first polypeptidechain and cysteine residue on the second polypeptide chain is not at theC-terminus of the second polypeptide chain.
 25. The diabody molecule ofclaim 24, wherein the covalent link between the first and second domainis a peptide bond.
 26. The diabody molecule of claim 24, wherein thecovalent link between the third and fourth domain is a peptide bond. 27.The diabody molecule of claim 24, wherein a disulfide bond is between atleast two cysteine residues on the first polypeptide chain and at leasttwo cysteine residues on the second polypeptide chain.
 28. The diabodymolecule of claim 1, wherein the first epitope and the second epitopeare different.
 29. The diabody molecule of claim 28, wherein the firstepitope and second epitope are epitopes from the same molecule.
 30. Thediabody molecule of claim 28, wherein the first epitope and secondepitope are epitopes from different molecules.
 31. The diabody moleculeof claim 1, wherein the first immunoglobulin or second immunoglobulin isa human or humanized immunoglobulin.
 32. The diabody molecule of claim31, wherein the first immunoglobulin or second immunoglobulin is a humanimmunoglobulin, which human immunoglobulin is an IgA, IgE, IgD, IgG orIgM.
 33. The diabody molecule of claim 32 wherein the humanimmunoglobulin is an IgG, which IgG is selected from the list consistingof IgG₁, IgG₂, IgG₃ and IgG₄.
 34. The diabody molecule of claim 1, 3, or10, wherein the Fc domain is a human Fc domain.
 35. The diabody moleculeof claim 34, wherein the Fc domain is an Fc domain of an IgA, IgE, IgD,IgG or IgM.
 36. The diabody molecule of claim 35, wherein the Fc domainis an Fc domain of an IgG, which IgG is selected from the listconsisting of IgG₁, IgG₂, IgG₃ or IgG₄.
 37. The diabody molecule ofclaim 1, wherein at least one epitope binding site is specific for aB-cell, T-cell, phagocytotic cell, natural killer (NK) cell or dendriticcell.
 38. The diabody molecule of claim 1, wherein at least one epitopebinding site is specific for a cell surface marker.
 39. The diabodymolecule of claim 38, wherein the cell surface marker is an Fc receptor.40. The diabody molecule of claim 39, wherein the Fc receptor is anactivating Fc receptor.
 41. The diabody molecule of claim 39, whereinthe Fc receptor is an inhibitory Fc receptor.
 42. The diabody moleculeof claim 39, wherein the Fc receptor is a Fcγ receptor. 43-64.(canceled)
 65. The diabody molecule of claim 42, wherein the secondepitope binding site is specific for a pathogenic antigen.
 66. Thediabody molecule of claim 65, wherein the pathogenic antigen is a tumorantigen, a bacterial antigen, viral antigen or autoimmune antigen. 67.The diabody molecule of claim 66, wherein the pathogenic antigen is abacterial antigen.
 68. The diabody molecule of claim 67, wherein thebacterial antigen is lipopolysaccharide.
 69. The diabody molecule ofclaim 66, wherein the pathogenic antigen is a viral antigen.
 70. Thediabody molecule of claim 69, wherein the viral antigen is selected fromthe group consisting of antigens from human immunodeficiency virus,Adenovirus and hepatitis virus.
 71. The diabody molecule of claim 66,wherein the pathogen antigen is an autoimmune antigen.
 72. The diabodymolecule of claim 71, wherein the autoimmune antigen is selected fromthe group consisting of DNA, RNA and collagen.
 73. The diabody moleculeof claim 38, wherein the second epitope binding site is specific for atoxin.
 74. The diabody molecule of claim 38, wherein the second epitopebinding site is specific for a drug.
 75. The diabody molecule of claim39, wherein the second epitope binding site is specific for a toxin. 76.The diabody molecule of claim 39, wherein the second epitope bindingsite is specific for a drug.
 77. The diabody molecule of claim 1,wherein the first epitope binding site is specific for CD32B and thesecond epitope binding site is specific for CD16A.
 78. The diabodymolecule of claim 3, wherein the first epitope binding site is specificfor CD16A and the second epitope binding site is specific for CD32B. 79.The diabody molecule of claim 4, wherein the first epitope binding siteis specific for CD16A and the second epitope binding site is specificfor CD32B. 80-88. (canceled)
 89. The diabody molecule of claim 1,wherein the third domain comprises a variant Fc region, which variant Fcregion comprises at least one amino acid modification relative to thewild type Fc region.
 90. The diabody molecule of claim 89, wherein saidmodification results in altered binding affinity between the Fc regionand an Fc receptor.
 91. The diabody molecule of claim 90, wherein saidmodification results in null binding between the Fc region and an Fcreceptor. 92-101. (canceled)
 102. A method for treating a disease ordisorder characterized by or associated with a pathogenic antigen, saidmethod comprising administering to a patient in need thereof aneffective amount of the diabody molecule of claim 66, which diabodymolecule binds to said pathogenic antigen.
 103. The method of claim 102wherein said disease or disorder is considered cancer.
 104. The methodof claim 102 wherein said disease or disorder is an infectious disease.105. A method for inducing immune tolerance to a pathogenic antigen,comprising administering to a patient in need thereof an effectiveamount of the diabody molecule of claim
 66. 106. A method fordetoxification comprising administering to a patient in need thereof aneffective amount of the diabody molecule of claim
 73. 107. A method fordetoxification comprising administering to a patient in need thereof aneffective amount of the diabody molecule of claim
 74. 108-113.(canceled)